De-immunized, shiga toxin a subunit scaffolds and cell-targeting molecules comprising the same

ABSTRACT

The present invention relates to Shiga toxin A Subunit derived polypeptides and cell-targeting molecules comprising amino acid substitutions which equip the polypeptides with 1) de-immunization; 2) reduced, protease-cleavage sensitivity; and/or 3) a heterologous epitope cargo(s) while retaining Shiga toxin function(s), such as, e.g., potent cytotoxicity. Certain polypeptides of the invention exhibit reduced immunogenic potential in mammals and/or are capable of delivering an epitope to an MHC class molecule of a cell in which the polypeptide is present. Certain molecules comprising a polypeptide of the invention are well-tolerated by mammals while retaining one or more of the features mentioned above. The Shiga toxin polypeptides of the invention have uses as components of cell-targeting molecules for selectively killing specific cells; for selectively delivering cargos to specific cells, and as therapeutic and/or diagnostic molecules for treating and diagnosing a variety of conditions, including cancers, immune disorders, and microbial infections.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/233,911, filed Apr. 19, 2021, which is a continuation of U.S.application Ser. No. 15/577,827, filed Nov. 29, 2017 (now abandoned),which is a national stage of International Application No.PCT/US2016/034778, filed on May 27, 2016, which claims priority to U.S.Provisional Application No. 62/168,758, filed on May 30, 2015, U.S.Provisional Application No. 62/168,759, filed on May 30, 2015, U.S.Provisional Application No. 62/168,760, filed on May 30, 2015, U.S.Provisional Application No. 62/168,761, filed on May 30, 2015, U.S.Provisional Application No. 62/168,762, filed on May 30, 2015, and U.S.Provisional Application No. 62/168,763, filed on May 30, 2015 thecontents of which are incorporated herein by reference in their entiretyfor all purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The content of the text file submitted electronically herewith isincorporated herein by reference in its entirety: A computer readableformat copy of the Sequence Listing (filename:MTEM_008_08US_SeqList_ST25.txt, date recorded: May 17, 2022, file size:about 939 kilobytes).

TECHNICAL FIELD

The present invention relates to Shiga toxin effector polypeptides,derived from the A Subunits of naturally occurring Shiga toxins, thatcomprise a combination of mutations providing (1) de-immunization, (2) areduction in protease sensitivity, and/or (3) an embedded, T-cellepitope(s); wherein the Shiga toxin effector polypeptides retain one ormore Shiga toxin functions, such as, e.g., potent cytotoxicity. Incertain embodiments, the Shiga toxin effector polypeptides of thepresent invention (1) exhibit reduced immunogenic potential in mammalsand/or (2) are each capable of delivering a CD8+ T-cell epitope to theMHC class I system of a cell in which the polypeptide is present.

The present invention also relates to cell-targeting molecules whichcomprise a Shiga toxin effector polypeptide of the present invention.The Shiga toxin effector polypeptides of the present invention have usesas scaffolds or components of cell-targeting molecules, such as, e.g.,immunotoxins and ligand-toxin fusions, for killing cells and/orsubcellular delivery of cargos to certain subcellular compartments, suchas, e.g., the delivery of an embedded, T-cell epitope to the cytosol. Ingeneral, the Shiga toxin effector polypeptides and cell-targetingmolecules of the present invention are useful for administration tomulticellular organisms, such as, e.g., when it is desirable to (1)eliminate or reduce non-specific toxicities, (2) eliminate or reducecertain immune responses, and/or (3) target a beneficial immuneresponse(s) to a specific epitope delivered to a specific cell-type,such as, e.g., the recruitment of CD8+ T-cells. The cell-targetingmolecules of the present invention are useful (1) for selectivelykilling specific cell type(s) amongst other cells and (2) as therapeuticmolecules for treating a variety of diseases, disorders, and conditions,including cancers, tumors, other growth abnormalities, immune disorders,and microbial infections.

BACKGROUND

The “magic bullet” concept is that therapeutics may be discovered thatspecifically attack only diseased cells or pathogens within a humanpatient while leaving the patient unharmed. Immunotoxins, ligand-toxinhybrids, immuno-RNases, and other molecularly targeted drugs aredescendants of Dr. Paul Ehrlich's “magic bullet” concept of the early20^(th) century (Strebhardt K, Ullrich A, Nat Rev Cancer 8: 473-80(2008)). The toxin produced by S. dysenteriae was named “Shiga toxin”after Dr. Ehrlich's associate Dr. Kiyoshi Shiga for his discovery ofthis bacterium in 1897. Recently, Shiga toxins have become appreciatedfor having unique characteristics favorable for use incell-internalizing molecules for targeted therapies (see e.g.US20150259428). Shiga toxins may be combined with immunoglobulindomains, ligands, and other targeting moieties to create cell-targetedtherapeutics (e.g., immunotoxins and ligand-toxin fusions) that are“magic bullets.”

Shiga toxins may have advantageous properties for use in therapeutics,such as, e.g., a potent toxin mechanism effective toward eukaryoticcells, ability to drive cellular internalization, and ability to directsubcellular routing. Shiga toxins have been synthetically engineered formedical applications by rational alterations to the toxin's structure,characteristics, and biological activities (see, e.g. WO 1999/040185, WO2005/092917, EP1051482, DE69835695, WO 2007/033497, US2009/0156417,JP4339511, U.S. Pat. No. 7,713,915, EP1727827, DE602004027168,EP1945660, JP4934761, EP2228383, US2013/0196928, WO 2014/164680, WO2014/164693, US20150259428, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, and WO 2015/191764, each of which isincorporated by reference herein in its entirety). Shiga toxin ASubunits are stable, enzymatically active, and cytotoxic even iftruncated or fused to other protein domains.

Major limitations to therapeutic applications involving syntheticallyengineered molecules derived from bacterial toxins include bothdetrimental immunogenic responses in recipients and non-specifictoxicities caused by toxic components. Unwanted immunogenicity of atherapeutic product could result in unfavorable consequences, such as areduced efficacy, the production of neutralizing antibodies, alteredpharmacokinetics, general immune and hypersensitivity reactions,anaphylaxis, anaphylactoid reactions, and constraints on the number ofrepeat doses a recipient can safely receive. Reducing the non-specifictoxicity of a therapeutic molecule can improve its safetycharacteristics when administered to a recipient as well as alter itspotential therapeutic window by increasing the maximum dosage which canbe administered safely. Because both unwanted immune responses andnon-specific toxicities can pose significant safety and/or efficacyissue(s) for a drug therapy, reducing or minimizing the probabilities ofboth is often desirable when developing therapeutic molecules.

The stability of a therapeutic or diagnostic molecule over time and inspecific environments (e.g. the human circulatory system) are importantfeatures and can affect for which applications a molecule may bepractically employed. For certain immunotoxins or ligand-toxin fusions,the stability of the linkage between the toxin and other components canaffect the amount of non-specific toxicity caused by the release ofuntargeted toxin over time within the body of a multicellular organism.Thus, for molecules comprising toxin components, certain non-specifictoxicities are directly related to the stability of the connectionbetween the toxin component and another component, such as, e.g., acell-targeting component.

Shiga toxins can be combined with heterologous epitopes to createcell-targeted therapeutics which deliver chosen epitope cargos for thepurpose of inducing desirable immune responses (see WO 2015/113007).These immune responses may be harnessed by therapeutic molecules for thetargeted killing of specific cell-types within a patient as well as tosensitize the patient's immune system to identifying certain cells asforeign (i.e. breaking immunotolerance). For example, the MajorHisto-Compatibility (MHC) class I presentation pathway may be exploitedby such approaches to induce the recruitment of immune cells to tumorloci within a patient and to enhance the recognition of certainneoplastic cells by immune surveillance mechanisms.

It would be desirable to have Shiga toxin A Subunit-derived polypeptideswith low antigenicity, low immunogenicity, and/or comprisingheterologous epitopes, but which retain a significant level of a Shigatoxin function(s), such as, e.g., potent cytotoxicity, the ability toforce cellular internalization, and/or the ability to efficiently routeto a desired intracellular location(s). Furthermore, it would bedesirable to have therapeutic and/or diagnostic molecules comprisingShiga toxin A Subunit derived components having low antigenicity, lowimmunogenicity, high stability, low non-specific toxicity, and/or theability to deliver peptide-epitope cargos for presentation by the MHCclass I system of a target cell. In particular, it would be desirable tohave cell-targeting molecules comprising Shiga toxin A Subunit derivedcomponents that maintain potent cytotoxicity while 1) reducing thepotential for unwanted antigenicities and/or immunogenicities, 2)reducing the potential for non-specific toxicities, and/or 3) having theability to deliver peptide-epitope cargos for presentation by the MHCclass I system of a target cell.

SUMMARY OF THE INVENTION

The Shiga toxin A Subunit derived scaffolds of the present inventioneach comprise a combination of features (e.g., de-immunizedsub-region(s), heterologous epitope comprising sub-region(s), aprotease-cleavage resistant sub-region, and/or a carboxy-terminal,endoplasmic reticulum retention/retrieval signal motif) which make themmore useful, such as, e.g., as components of cell-targeting moleculeslike immunotoxins and ligand-toxin fusions. Certain combination Shigatoxin effector polypeptides of the present invention are more usefulbecause they provide several Shiga toxin effector functions in a singlepolypeptide, such as, e.g., promoting cellular internalization,directing sub-cellular routing to the cytosol, ribosome inactivation,and/or delivering heterologous, T-cell epitopes to the MHC I classpathway of a cell. Certain cell-targeting molecules of the presentinvention are more useful because they provide a combination of severalproperties in a single molecule, such as, e.g., efficient cellularinternalization, potent cell-targeted cytotoxicity, selectivecytotoxicity, de-immunization, low non-specific toxicity at highdosages, high stability, CD+ T-cell hyper-immunization, and/or theability to deliver a heterologous, T-cell epitope(s) to the MHC I classpathway of a target cell.

Different embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are described belowwith reference to sets of embodiments numbered #1-11.

Embodiment Set #1—De-Immunized, Shiga Toxin Effector PolypeptideComprising an Embedded or Inserted, Heterologous, T-Cell Epitope

The present invention provides a de-immunized, Shiga toxin effectorpolypeptide comprising at least one inserted or embedded, heterologousepitope (a) and at least one disrupted, endogenous, B-cell and/or CD4+T-cell epitope region (b), wherein the heterologous epitope does notoverlap with at least one disrupted, endogenous, B-cell and/or CD4+T-cell epitope region; and wherein the Shiga toxin effector polypeptideis capable of exhibiting at least one Shiga toxin effector function (seee.g. FIG. 1, depicting illustrative examples of three, exemplaryembodiments of the de-immunized Shiga toxin effector polypeptide of thisembodiment set #1 labeled Shiga toxin effector 1, 2, and 3). In certainfurther embodiments, the heterologous epitope is a CD8+ T-cell epitopecapable of being presented by a MHC class I molecule of a cell. Incertain further embodiments, the heterologous epitope in (a) is embeddedand replaces an equivalent number of amino acid residues in a wild-typeShiga toxin polypeptide region such that the Shiga toxin effectorpolypeptide has the same total number of amino acid residues as does thewild-type Shiga toxin polypeptide region from which it is derived. Incertain further embodiments of any of the above, the de-immunized, Shigatoxin effector polypeptide is capable of exhibiting at least one Shigatoxin effector function selected from: directing intracellular routingto a cytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, andcytotoxicity.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises (i) an embedded orinserted, heterologous, T-cell epitope and (ii) a disruption of at leastone, endogenous, B-cell and/or T-cell epitope which does not overlapwith the embedded or inserted, heterologous, T-cell epitope. In certainfurther embodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. In certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the endogenous, T-cell epitope is a CD4+ T-cell epitope,such as, e.g., with regard to a human immune system.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises (i) an embedded orinserted, heterologous, T-cell epitope and (ii) a disruption of at leastone, endogenous, B-cell and/or T-cell epitope region which does notoverlap with the embedded or inserted, heterologous, T-cell epitope. Incertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. In certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the endogenous, T-cell epitope region is a CD4+ T-cellepitope region, such as, e.g., with regard to a human immune system.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises (i) an embedded orinserted, heterologous, T-cell epitope and (ii) a disruption of at leastone, endogenous, B-cell and/or T-cell epitope which does not overlapwith the embedded or inserted, heterologous, T-cell epitope. In certainfurther embodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. In certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the endogenous, T-cell epitope is a CD4+ T-cell epitope,such as, e.g., with regard to a human immune system.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises (i) an embedded orinserted, heterologous, T-cell epitope and (ii) a disruption of at leastone, endogenous, B-cell and/or T-cell epitope region which does notoverlap with the embedded or inserted, heterologous, T-cell epitope. Incertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. In certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the endogenous, T-cell epitope region is a CD4+ T-cellepitope region, such as, e.g., with regard to a human immune system.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218 of SEQ ID NO:3;240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ IDNO:3; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO:1 or SEQID NO:2; 34-78 of SEQ ID NO:1 or SEQ ID NO:2; 77-103 of SEQ ID NO:1 orSEQ ID NO:2; 128-168 of SEQ ID NO:1 or SEQ ID NO:2; 160-183 of SEQ IDNO:1 or SEQ ID NO:2; 236-258 of SEQ ID NO:1 or SEQ ID NO:2; and 274-293of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxinA Subunit or derivative thereof. In certain further embodiments, thereis no disruption which is a carboxy-terminal truncation of amino acidresidues that overlap with part or all of at least one disrupted,endogenous, B-cell and/or T-cell epitope and/or epitope region.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell immunogenic, amino acid residue selected from thegroup of natively positioned Shiga toxin A Subunit amino acid residues:L49, D197, D198, R204, and R205.

In certain embodiments of Embodiment Set #1, the embedded or inserted,heterologous, T-cell epitope disrupts the endogenous, B-cell and/orT-cell epitope region is selected from the group of natively positionedShiga toxin A Subunit regions consisting of: (i) 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO: 3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or theequivalent region in a Shiga toxin A Subunit or derivative thereof,wherein there is no disruption which is an amino-terminal truncation ofsequences that overlap with part or all of at least one disruptedepitope region; (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141-153 of SEQ ID NO:1 or SEQ ID NO: 2; 140-156 of SEQ ID NO:3; 179-190of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO: 3;and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2;254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent region in a Shiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: (i) 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ IDNO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; or the equivalent region in a Shiga toxin ASubunit or derivative thereof, wherein there is no disruption which isan amino-terminal truncation of sequences that overlap with part or allof at least one disrupted epitope region; (ii) 94-115 of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO: 2;140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2;and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 ofSEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2;262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ IDNO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxin A Subunitor derivative thereof, wherein there is no disruption which is anamino-terminal truncation of sequences that overlap with part or all ofat least one disrupted epitope region.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a disruption of at least one endogenous epitoperegion selected from the group of natively positioned Shiga toxin ASubunits consisting of: 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3;179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 ofSEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; or 210-218 of SEQ IDNO:3.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide does not comprise a heterologous, MHC class I-restricted,T-cell epitope. MHC class I-restricted, T-cell epitopes are known in theart or can be predicted by the skilled worker. The term heterologousrefers to MHC class I-restricted, T-cell epitopes which are not nativelypresent in wild-type Shiga toxin A Subunits, such as, e.g., thewild-type Shiga toxin A Subunit which is most closely related to theShiga toxin effector polypeptide of interest.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises disruptions of at least two, three, four, five,six, seven, eight, or more endogenous, B-cell and/or T-cell epitoperegions.

In certain embodiments of Embodiment Set #1, one or more disruptionscomprises an amino acid residue substitution relative to a wild-typeShiga toxin A Subunit.

In certain embodiments of Embodiment Set #1, one or more endogenous,B-cell and/or T-cell epitope regions comprises a plurality of amino acidresidue substitutions relative to a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Set #1, at least one, two, three,or four disruptions comprise a plurality of amino acid residuesubstitutions in the endogenous, B-cell and/or T-cell epitope regionrelative to a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Set #1, at least one disruptioncomprises at least one, two, three, four, five, six, seven, eight, ormore amino acid residue substitutions relative to a wild-type Shigatoxin A Subunit, and optionally wherein at least one substitution occursat the natively positioned Shiga toxin A Subunit amino acid residueselected form the group consisting of: 1 of SEQ ID NO:1 or SEQ ID NO:2;4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 9of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO:3; 12 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 ofSEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQ ID NO:1 or SEQ ID NO:2;48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ IDNO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 57 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO: 3; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 60 of SEQ ID NO:1 orSEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 96of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2;111 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 112 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ IDNO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO:3;180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2;183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2;188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2;197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ IDNO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQID NO:1 or SEQ ID NO:2; or the equivalent amino acid residue in a Shigatoxin A Subunit or derivative thereof. In certain further embodiments,at least two disruptions each comprise at least one amino acid residuesubstitutions relative to a wild-type Shiga toxin A Subunit selectedform the group consisting of: 1 of SEQ ID NO:1 or SEQ ID NO:2; 4 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 47 ofSEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ IDNO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 58 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 ofSEQ ID NO:1 or SEQ ID NO: 2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQID NO:1 or SEQ ID NO:2; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;96 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 109 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO: 3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 112 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 ofSEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 ofSEQ ID NO:1, SEQ ID SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ IDNO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ IDNO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 250 of SEQ ID NO:3; 264 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ IDNO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent aminoacid residue in a Shiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises disruption of at least three, endogenous, B-celland/or T-cell epitope regions selected from the group of consisting of:(i) 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3, or the equivalent region in a Shiga toxin ASubunit or derivative thereof, wherein there is no disruption which isan amino-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope region; (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; and(iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2;254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; or theequivalent region in a Shiga toxin A Subunit or derivative thereof,wherein there is no disruption which is a carboxy-terminal truncation ofamino acid residues that overlap with part or all of at least onedisrupted, endogenous, B-cell and/or T-cell epitope and/or epitoperegion.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises disruptions of at least two, endogenous, B-celland/or T-cell epitope regions, wherein each disruption comprises one ormore amino acid residue substitutions, and wherein the endogenous,B-cell and/or T-cell epitope regions are selected from the group ofnatively positioned Shiga toxin A Subunit regions consisting of: 3-14 ofSEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2;39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; or the equivalent region in aShiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Set #1, the embedded or inserted,heterologous, T-cell epitope does not disrupt any endogenous, B-celland/or CD4+ T-cell epitope region described herein.

In certain embodiments of Embodiment Set #1, at least one disruptioncomprises one or more amino acid residue substitutions relative to awild-type Shiga toxin A Subunit is selected from the group consistingof: D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, D toM, D to R, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E toQ, E to N, E to D, E to M, E to R, F to A, F to G, F to V, F to L, F toI, G to A, G to P, H to A, H to G, H to V, H to L, H to I, H to F, H toM, I to A, I to V, I to G, I to C, K to A, K to G, K to V, K to L, K toI, K to M, K to H, L to A, L to V, L to G, L to C, N to A, N to G, N toV, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R toV, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S toA, S to G, S to V, S to L, S to I, S to F, S to M, T to A, T to G, T toV, T to L, T to I, T to F, T to M, T to S, V to A, V to G, Y to A, Y toG, Y to V, Y to L, Y to I, Y to F, Y to M, and Y to T. In certainfurther embodiments, the one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, Eto N, E to D, E to M, E to R, G to A, H to A, H to G, H to V, H to L, Hto I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, Kto H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, Pto A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, Rto M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, Sto I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, Tto M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, and Y toM.

In certain embodiments of Embodiment Set #1, at least one of thedisruption(s) comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit selected from the groupconsisting of: K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F,M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F,and M; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M;K11 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K;S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, andM; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I,F, S, M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; Y49to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51; D53 toA, G, V, L, I, F, S, and Q; V54 to A, G, I, and L; R55 to A, G, V, L, I,F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A,V, C, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, andF; E60 to A, G, V, L, I, F, S, Q, N, D, M, T, and R; E61 to A, G, V, L,I, F, S, Q, N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S,K, and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I,F, S, and Q; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F,M; and N; A105 to L; T107 to A, G, V, L, I, F, M, and P; S107 to A, G,V, L, I, F, M, and P; L108 to A, V, C, and G; S109 to A, G, V, I, L, F,and M; T109 to A, G, V, I, L, F, M, and S; G110 to A; S112 to A, G, V,L, I, F, and M; D111 to A, G, V, L, I, F, S, Q, and T; S112 to A, G, V,L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to Aand G. R179 to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L,I, F, M, and S; T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L,I, F, S, and Q; D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, V andC; S186 to A, G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F,M, Q, S, K, and H; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L,I, F, S, and Q; D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L,I, F, M, Q, S, K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H;S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R248to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q,S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G,V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide consists essentially of the polypeptide shown in any one ofSEQ ID NOs: 355-369 which further comprises a disruption of at leastone, endogenous, B-cell and/or T-cell epitope region which does notoverlap with an embedded or inserted, heterologous, CD8+ T-cell epitope.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises or consists essentially of the polypeptide shownin any one of SEQ ID NOs: 6-32, 340-354, and 370-438.

For certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide is capable of exhibiting (i) a catalytic activity levelcomparable to a wild-type Shiga toxin A1 fragment or wild-type Shigatoxin effector polypeptide, (ii) a ribosome inhibition activity with anhalf-maximal inhibitory concentration (IC₅₀) value of 10,000 picomolaror less, and/or (iii) a significant level of Shiga toxin catalyticactivity.

For certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide is capable of exhibiting subcellular routing efficiencycomparable to a wild-type Shiga toxin effector polypeptide and/orcapable of exhibiting a significant level of intracellular routingactivity to the endoplasmic reticulum and/or cytosol from an endosomalstarting location of a cell.

For certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide is capable of intracellular delivery of the embedded orinserted, heterologous, T-cell epitope from an early endosomalcompartment to a MHC class I molecule of a cell in which the Shiga toxineffector polypeptide is present. For certain further embodiments, theShiga toxin effector polypeptide is capable of exhibiting one or moreShiga toxin effector functions in addition to the intracellulardelivery, such as, e.g., the Shiga toxin effector functions of:promoting cellular internalization, directing sub-cellular routing tothe cytosol, ribosome inactivation, inducing caspase activity, causingcytostasis, and/or causing cell death. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a disruption of at least two, endogenous, epitoperegions selected the group of natively positioned Shiga toxin A Subunitregions consisting of: 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3;179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 ofSEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; or 210-218 of SEQ IDNO:3; wherein the disruption does not consist solely of the amino acidresidue substitution selected from the group consisting of: S96Y of SEQID NO:1 or SEQ ID NO:2; Y114S of SEQ ID NO:1 or SEQ ID NO:2; R179A ofSEQ ID NO:1 or SEQ ID NO:2; R179H of SEQ ID NO:1 or SEQ ID NO:2; L185Aof SEQ ID NO:1 or SEQ ID NO:2; R188A of SEQ ID NO:1 or SEQ ID NO:2;R205A of SEQ ID NO:1 or SEQ ID NO:2; R179A/R188A of SEQ ID NO:1; or SEQID NO:2; or A188V of SEQ ID NO:3.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a disruption comprising a mutation of at least oneamino acid residue in at least one, endogenous epitope region selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3;26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; wherein the Shiga toxin effector polypeptidecomprises no amino-terminus truncation overlapping with theaforementioned disrupted epitope region.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a disruption comprising a mutation of at least oneamino acid residue in at least two, endogenous epitope regions selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3;26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; wherein the disruption does not consist solelyof the amino acid residue substitution R63W of SEQ ID NO:1 or SEQ IDNO:2; and wherein the Shiga toxin effector region comprises noamino-terminus truncation overlapping with the aforementioned, two,disrupted epitope regions.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a disruption comprising a mutation of at least oneamino acid residue in at least one, endogenous epitope region selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ IDNO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3;281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2;wherein the Shiga toxin effector polypeptide comprises nocarboxy-terminus truncation overlapping with the aforementioneddisrupted epitope region.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises a disruption comprising a mutation of at least oneamino acid residue in at least two, endogenous epitope regions selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ IDNO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3;281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2;wherein the Shiga toxin effector polypeptide does not comprise themutation selected from the group consisting of: R248H of SEQ ID NO:1 orSEQ ID NO:2; A250V of SEQ ID NO:1 or SEQ ID NO:2; R251H of SEQ ID NO:1or SEQ ID NO:2; A253G of SEQ ID NO:1 or SEQ ID NO:2; S254T of SEQ IDNO:1 or SEQ ID NO:2; C261A of SEQ ID NO:1 or SEQ ID NO:2; R289K of SEQID NO:1 or SEQ ID NO:2; R248H and R251H of SEQ ID NO:1 or SEQ ID NO:2;A253G and S254T of SEQ ID NO:1 or SEQ ID NO:2; the deletion of S247-M252of SEQ ID NO:1; S246F of SEQ ID NO:3; A282V of SEQ ID NO:3; 1291V of SEQID NO:3; S246F of SEQ ID NO:3; and wherein the Shiga toxin effectorpolypeptide comprises no carboxy-terminus truncation overlapping withthe aforementioned, two, disrupted epitope regions.

In certain embodiments of Embodiment Set #1, the de-immunized, Shigatoxin effector polypeptide comprises or consists essentially of thepolypeptide shown in any one of SEQ ID NOs: 6-27, 29-32, 340-354, and370-438.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide of the present invention comprises one or more mutationsrelative to a naturally occurring A Subunit of a member of the Shigatoxin family which changes an enzymatic activity of the Shiga toxineffector polypeptide, the mutation selected from at least one amino acidresidue deletion, insertion, or substitution. In certain furtherembodiments, the mutation relative to the naturally occurring A Subunitreduces of eliminates a cytotoxic activity of the Shiga toxin effectorpolypeptide but the Shiga toxin effector polypeptide retains at leastone other Shiga toxin effector function, such as, e.g., promotingcellular internalization and/or directing intracellular routing to acertain subcellular compartment(s). In certain further embodiments, themutation relative to the naturally occurring A Subunit is selected fromat least one amino acid residue substitution, such as, e.g., A231E,R75A, Y77S, Y114S, E167D, R170A, R176K, and/or W203A in SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises (i) a Shiga toxin A1 fragment derived regionhaving a carboxy terminus and (ii) a disrupted furin-cleavage motif atthe carboxy-terminus of the A1 fragment region. In certain furtherembodiments, the disrupted furin-cleavage motif comprises one or moremutations, relative to a wild-type Shiga toxin A Subunit, the mutationaltering at least one amino acid residue in a region natively positionedat 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ ID NO: 1) orShiga toxin (SEQ ID NO:2), or at 247-250 of the A Subunit of Shiga-liketoxin (SEQ ID NO:3); or the equivalent region in a Shiga toxin A Subunitor derivative thereof. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, in a minimal furin cleavage site of thefurin-cleavage motif. In certain further embodiments the minimal furincleavage site is represented by the consensus amino acid sequenceR/Y-x-x-R and/or R-x-x-R.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide comprises (i) a Shiga toxin A1 fragment derived regionhaving a carboxy terminus and (ii) a disrupted furin-cleavage motif atthe carboxy-terminus of the A1 fragment region. In certain furtherembodiments, the disrupted furin-cleavage motif comprises an amino acidresidue substitution in the furin-cleavage motif relative to a wild-typeShiga toxin A Subunit. In certain further embodiments, the substitutionof the amino acid residue in the furin-cleavage motif is of an arginineresidue with a non-positively charged, amino acid residue selected fromthe group consisting of: alanine, glycine, proline, serine, threonine,aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine,leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine. Incertain embodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide, either alone or as a component of a first cell-targetingmolecule, is capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a wild-type Shiga toxin A1 polypeptideand/or second cell-targeting molecule consisting of the firstcell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment.

For certain embodiments of the Shiga toxin effector polypeptide ofEmbodiment Set #1, a cell-targeting molecule of the present inventioncomprising the Shiga toxin effector polypeptide is capable whenintroduced to a chordate of exhibiting improved, in vivo tolerabilitycompared to a second cell-targeting molecule consisting of the firstcell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment and/or wild-type Shiga toxin furin-cleavage site at the carboxyterminus of its A1 fragment region.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide of the present invention comprises a Shiga toxin effectorregion derived from amino acids 75 to 251 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3.

In certain embodiments of Embodiment Set #1, the Shiga toxin effectorpolypeptide of the present invention comprises a Shiga toxin effectorregion derived from amino acids 1 to 241 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3. In certain further embodiments, the Shiga toxin effectorregion is derived from amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3. In certain further embodiments, the Shiga toxin effectorregion is derived from amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3.

Embodiment Set #2—Cell-Targeting Molecule Comprising a De-immunizedShiga Toxin Effector Polypeptide Comprising an Embedded or Inserted,Heterologous, T-Cell Epitope and A Non-overlapping De-ImmunizedSub-Region

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a de-immunized, Shiga toxin effectorpolypeptide of Embodiment Set #1 (see e.g. FIG. 1, depictingillustrative examples of four, exemplary embodiments of thecell-targeting molecules of this embodiment set #2). For example,certain embodiments of set #2 is the cell-targeting molecule comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a de-immunized, Shiga toxin effectorpolypeptide comprising at least one inserted or embedded, heterologousepitope (a) and at least one disrupted, endogenous, B-cell and/or CD4+T-cell epitope region (b), wherein the heterologous epitope does notoverlap with the embedded or inserted, heterologous, T-cell epitope. Forcertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface. For certainfurther embodiments, the cell-targeting molecule is capable whenintroduced to cells of exhibiting a cytotoxicity comparable or betterthan a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention is capable when introduced to achordate of exhibiting improved in vivo tolerability and/or stabilitycompared to a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide component(s) each comprise a wild-typeShiga toxin A1 fragment and/or wild-type Shiga toxin furin-cleavage siteat the carboxy terminus of its A1 fragment region. In certain furtherembodiments, the Shiga toxin effector polypeptide is not cytotoxic andthe molecular moiety is cytotoxic.

In certain embodiments of Embodiment Set #2, the binding region andShiga toxin effector polypeptide are linked together, either directly orindirectly.

In certain embodiments of Embodiment Set #2, the binding regioncomprises a polypeptide comprising an immunoglobulin-type bindingregion. In certain further embodiments, the binding region comprising apolypeptide selected from the group consisting of: an autonomous V_(H)domain, single-domain antibody fragment (sdAb), nanobody, heavychain-antibody domain derived from a camelid (V_(H)H or V_(H) domainfragment), heavy-chain antibody domain derived from a cartilaginous fish(V_(H)H or V_(H) domain fragment), immunoglobulin new antigen receptor(IgNAR), V_(NAR) fragment, single-chain variable fragment (scFv),antibody variable fragment (Fv), complementary determining region 3fragment (CDR3), constrained FR3-CDR3-FR4 polypeptide (FR3-CDR3-FR4), Fdfragment, small modular immunopharmaceutical (SMIP) domain,antigen-binding fragment (Fab), Armadillo repeat polypeptide (ArmRP),fibronectin-derived 10^(th) fibronectin type III domain (10Fn3),tenascin type III domain (TNfn3), ankyrin repeat motif domain,low-density-lipoprotein-receptor-derived A-domain (LDLR-A), lipocalin(anticalin), Kunitz domain, Protein-A-derived Z domain, gamma-Bcrystalline-derived domain, ubiquitin-derived domain, Sac7d-derivedpolypeptide (affitin), Fyn-derived SH2 domain, miniprotein, C-typelectin-like domain scaffold, engineered antibody mimic, and anygenetically manipulated counterparts of any of the foregoing whichretain binding functionality.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a activitywith a half-maximal inhibitory concentration (IC₅₀) value of 10,000picomolar or less, and/or (iii) a significant level of Shiga toxincatalytic activity.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention and/or its Shiga toxin effectorpolypeptide is capable of exhibiting subcellular routing efficiencycomparable to a reference cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment or wild-type Shiga toxin effector polypeptideand/or capable of exhibiting a significant level of intracellularrouting activity to the endoplasmic reticulum and/or cytosol from anendosomal starting location of a cell.

For certain embodiments of Embodiment Set #2, whereby administration ofthe cell-targeting molecule of the present invention to a cellphysically coupled with the extracellular target biomolecule of thecell-targeting molecule's binding region, the cell-targeting molecule iscapable of causing death of the cell. In certain further embodiments,administration of the cell-targeting molecule of the invention to twodifferent populations of cell types which differ with respect to thepresence or level of the extracellular target biomolecule, thecell-targeting molecule is capable of causing cell death to thecell-types physically coupled with an extracellular target biomoleculeof the cytotoxic cell-targeting molecule's binding region at a CD₅₀ atleast three times or less than the CD₅₀ to cell types which are notphysically coupled with an extracellular target biomolecule of thecell-targeting molecule's binding region. For certain embodiments,whereby administration of the cell-targeting molecule of the presentinvention to a first populations of cells whose members are physicallycoupled to extracellular target biomolecules of the cell-targetingmolecule's binding region, and a second population of cells whosemembers are not physically coupled to any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationsof cells whose members are physically coupled to a significant amount ofthe extracellular target biomolecule of the cell-targeting molecule'sbinding region, and a second population of cells whose members are notphysically coupled to a significant amount of any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationof target biomolecule positive cells, and a second population of cellswhose members do not express a significant amount of a targetbiomolecule of the cell-targeting molecule's binding region at acellular surface, the cytotoxic effect of the cell-targeting molecule tomembers of the first population of cells relative to members of thesecond population of cells is at least 3-fold greater.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity.

For certain embodiments of Embodiment Set #2, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculecomprises a molecular moiety associated with the carboxy-terminus of theShiga toxin effector polypeptide. In certain embodiments, the molecularmoiety comprises or consists of the binding region. In certainembodiments, the molecular moiety comprises at least one amino acid andthe Shiga toxin effector polypeptide is linked to at least one aminoacid residue of the molecular moiety. In certain further embodiments,the molecular moiety and the Shiga toxin effector polypeptide are fusedforming a continuous polypeptide.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculefurther comprises a cytotoxic molecular moiety associated with thecarboxy-terminus of the Shiga toxin effector polypeptide. For certainembodiments, the cytotoxic molecular moiety is a cytotoxic agent, suchas, e.g., a small molecule chemotherapeutic agent, anti-neoplasticagent, cytotoxic antibiotic, alkylating agent, antimetabolite,topoisomerase inhibitor, and/or tubulin inhibitor known to the skilledworker and/or described herein. For certain further embodiments, thecytotoxic molecular moiety is cytotoxic at concentrations of less than10,000, 5,000, 1,000, 500, or 200 pM.

In certain embodiments of Embodiment Set #2, the binding region iscapable of binding to an extracellular target biomolecule selected fromthe group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30,HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen,Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19,CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72,tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1),carbonic anhydrase IX, folate binding protein, ganglioside GD2,ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, AlphaVbeta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1,TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA,gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE,NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6,SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen,prostate stem cell antigen, human aspartyl (asparaginyl)beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100,tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen,Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38,CD15, CD23, CD45 (protein tyrosine phosphatase receptor type C), CD53,CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa,galectin-9, IL-1R (interleukin-1 receptor), mrp-14, NKG2D ligand,programmed death-ligand 1 (PD-L1), Siglec-8, Siglec-10, CD49d, CD13,CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14,CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c,GITRL, MHC class I molecule, MHC class II molecule (optionally complexedwith a peptide), CD284 (TLR4), CD107-Mac3, CD195 (CCR5), HLA-DR,CD16/32, CD282 (TLR2), CD11c, and any immunogenic fragment of any of theforegoing.

In certain embodiments of Embodiment Set #2, the binding region islinked, either directly or indirectly, to the Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, the binding region is fused, eitherdirectly or indirectly, to the carboxy-terminus of the Shiga toxineffector polypeptide to form a single, continuous polypeptide. Incertain further embodiments, the binding region is animmunoglobulin-type binding region.

In certain embodiments of Embodiment Set #2, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site. Incertain embodiments, the mutation in the minimal, furin-cleavage site isan amino acid deletion, insertion, and/or substitution of at least oneamino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned 1) at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ IDNO: 1) or Shiga toxin (SEQ ID NO: 2), or 2) at 247-250 of the A Subunitof Shiga-like toxin 2 (SEQ ID NO:3), or the equivalent amino acidsequence position in any Shiga toxin A Subunit. In certain furtherembodiments, the mutation is an amino acid residue substitution of anarginine residue with a non-positively charged, amino acid residue.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity comparable to a cytotoxicity of a referencemolecule, such as, e.g., a second cell-targeting molecule consisting ofthe cell-targeting molecule except for all of its Shiga toxin effectorpolypeptide component(s) each comprise a wild-type Shiga toxin A1fragment.

In certain embodiments of Embodiment Set #2, the binding regioncomprises the peptide or polypeptide shown in any one of SEQ ID NOs:83-339. In certain further embodiments, the binding region comprises orconsists essentially of the polypeptide represented by any of thefollowing: amino acids 1-245 of any one of SEQ ID NOs: 33, 64, and 65;269-513 of SEQ ID NO:40 or SEQ ID NO:80; amino acids 269-520 or 269-521of any one of SEQ ID NOs: 36, 66, and 67; amino acids 1-232, 1-233,1-234, 1-235, 1-236, 1-242, 1-243, 1-244, 1-245, 1-246, 1-252, 1-253,1-254, 1-255, or 1-256 of any one of SEQ ID NOs: 47-119 and 176-248;amino acids 269-498 or 269-499 of any one of SEQ ID NOs: 37-39, 68-79,and 81; amino acids 269-499, 269-512, 269-513, or 280-510 of any one ofSEQ ID NOs: 34, 35, 41-56, and 82.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention comprises or consists essentially of thepolypeptide shown in any one of SEQ ID NOs: 43-62, 64-82, and 439-513.

In certain embodiments of Embodiment Set #2, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #2, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #2, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #2, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is capable when introduced to cells of exhibiting cytotoxicitythat is greater than that of a third cell-targeting molecule having anamino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. For certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of the third cell-targeting molecule. Forcertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the third cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.In certain further embodiments, the third cell-targeting molecule doesnot comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family (“KDEL” disclosed asSEQ ID NO: 514).

In certain embodiments of Embodiment Set #2, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a thirdcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide which is notpositioned at or proximal to the amino-terminus of the thirdcell-targeting molecule. In certain further embodiments, the thirdcell-targeting molecule does not comprise any carboxy-terminal,endoplasmic reticulum retention/retrieval signal motif of the KDELfamily.

In certain embodiments of Embodiment Set #2, the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention exhibits low cytotoxic potency (i.e. is not capable whenintroduced to certain positive target cell types of exhibiting acytotoxicity greater than 1% cell death of a cell population at acell-targeting molecule concentration of 1000 nM, 500 nM, 100 nM, 75 nM,or 50 nM) and is capable when introduced to cells of exhibiting agreater subcellular routing efficiency from an extracellular space to asubcellular compartment of an endoplasmic reticulum and/or cytosol ascompared to the cytotoxicity of a third cell-targeting molecule havingan amino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. In certain furtherembodiments, the third cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Set #2, the cell-targeting moleculeof the present invention, or a polypeptide component thereof, comprisesa carboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif of a member of the KDEL family. For certain further embodiments,the carboxy-terminal endoplasmic reticulum retention/retrieval signalmotif is selected from the group consisting of:

(SEQ ID NO: 514) KDEL, (SEQ ID NO: 561) HDEF, (SEQ ID NO: 515) HDEL,(SEQ ID NO: 562) RDEF, (SEQ ID NO: 516) RDEL, (SEQ ID NO: 517) WDEL,(SEQ ID NO: 518) YDEL, (SEQ ID NO: 563) HEEF, (SEQ ID NO: 519) REEL,(SEQ ID NO: 520) KEEL, (SEQ ID NO: 521) REEL, (SEQ ID NO: 564) KAEL,(SEQ ID NO: 565) KCEL, (SEQ ID NO: 522) KFEL, (SEQ ID NO: 566) KGEL,(SEQ ID NO: 567) KHEL, (SEQ ID NO: 568) KLEL, (SEQ ID NO: 569) KNEL,(SEQ ID NO: 570) KQEL, (SEQ ID NO: 571) KREL, (SEQ ID NO: 572) KSEL,(SEQ ID NO: 573) KVEL, (SEQ ID NO: 574) KWEL, (SEQ ID NO: 575) KYEL,(SEQ ID NO: 576) KEDL, (SEQ ID NO: 523) KIEL, (SEQ ID NO: 524) DKEL,(SEQ ID NO: 577) FDEL, (SEQ ID NO: 578) KDEF, (SEQ ID NO: 525) KKEL,(SEQ ID NO: 579) HADL, (SEQ ID NO: 580) HAEL, (SEQ ID NO: 581) HIEL,(SEQ ID NO: 526) HNEL, (SEQ ID NO: 527) HTEL, (SEQ ID NO: 528) KTEL,(SEQ ID NO: 529) HVEL, (SEQ ID NO: 582) NDEL, (SEQ ID NO: 583) QDEL,(SEQ ID NO: 584) REDL, (SEQ ID NO: 585) RNEL, (SEQ ID NO: 586) RTDL,(SEQ ID NO: 587) RTEL, (SEQ ID NO: 588) SDEL, (SEQ ID NO: 589) TDEL,(SEQ ID NO: 590) SKEL, (SEQ ID NO: 591) STEL, and (SEQ ID NO: 592) EDEL.In certain further embodiments, the cell-targeting molecule of thepresent invention is capable when introduced to cells of exhibitingcytotoxicity that is greater than that of a fourth cell-targetingmolecule consisting of the cell-targeting molecule except for it doesnot comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to a reference molecule, such as, e.g., thefourth cell-targeting molecule. In certain further embodiments, thecytotoxicity of the cell-targeting molecule of the present invention toa population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of thefourth cell-targeting molecule to a second population of target positivecells as assayed by CD₅₀ values.

Embodiment Set #3—Cell-Targeting Molecule Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and a Shiga ToxinEffector Polypeptide Comprising an Embedded or Inserted, Heterologous,T-Cell Epitope

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprisingan inserted or embedded, heterologous, epitope; and (iii) acarboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (a) a binding region capable of specificallybinding at least one extracellular target biomolecule; (b) a Shiga toxineffector polypeptide comprising an embedded or inserted, heterologousepitope; and (c) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of a member of the KDEL family. Forcertain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface.

In certain embodiments of Embodiment Set #3, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL (SEQ ID NO: 514), HDEF (SEQ ID NO: 561),HDEL (SEQ ID NO: 515), RDEF (SEQ ID NO: 562), RDEL (SEQ ID NO: 516),WDEL (SEQ ID NO: 517), YDEL (SEQ ID NO: 518), HEEF (SEQ ID NO: 563),HEEL (SEQ ID NO: 519), KEEL (SEQ ID NO: 520), REEL (SEQ ID NO: 521),KAEL (SEQ ID NO: 564), KCEL (SEQ ID NO: 565), KFEL (SEQ ID NO: 522),KGEL (SEQ ID NO: 566), KHEL (SEQ ID NO: 567), KLEL (SEQ ID NO: 568),KNEL (SEQ ID NO: 569), KQEL (SEQ ID NO: 570), KREL (SEQ ID NO: 571),KSEL (SEQ ID NO: 572), KVEL (SEQ ID NO: 573), KWEL (SEQ ID NO: 574),KYEL (SEQ ID NO: 575), KEDL (SEQ ID NO: 576), KIEL (SEQ ID NO: 523),DKEL (SEQ ID NO: 524), FDEL (SEQ ID NO: 577), KDEF (SEQ ID NO: 578),KKEL (SEQ ID NO: 525), HADL (SEQ ID NO: 579), HAEL (SEQ ID NO: 580),HIEL (SEQ ID NO: 581), HNEL (SEQ ID NO: 526), HTEL (SEQ ID NO: 527),KTEL (SEQ ID NO: 528), HVEL (SEQ ID NO: 529), NDEL (SEQ ID NO: 582),QDEL (SEQ ID NO: 583), REDL (SEQ ID NO: 584), RNEL (SEQ ID NO: 585),RTDL (SEQ ID NO: 586), RTEL (SEQ ID NO: 587), SDEL (SEQ ID NO: 588),TDEL (SEQ ID NO: 589), SKEL (SEQ ID NO: 590), STEL (SEQ ID NO: 591), andEDEL (SEQ ID NO: 592).

In certain embodiments of Embodiment Set #3, the embedded or inserted,heterologous, T-cell epitope disrupts the endogenous, B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: (i) 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or theequivalent region in a Shiga toxin A Subunit or derivative thereof, (ii)94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ IDNO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 orSEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO: 3; 205 of SEQ IDNO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO: 3; and (iii) 240-260 ofSEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ IDNO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and285-293 of SEQ ID NO:1 or SEQ ID NO:2, or the equivalent region in aShiga toxin A Subunit or derivative thereof.

In certain further embodiments of Embodiment Set #3, the heterologousepitope is a CD8+ T-cell epitope capable of being presented by a MHCclass I molecule of a cell. In certain further embodiments, theheterologous epitope in is embedded and replaces an equivalent number ofamino acid residues in a wild-type Shiga toxin polypeptide region suchthat the Shiga toxin effector polypeptide has the same total number ofamino acid residues as does the wild-type Shiga toxin polypeptide regionfrom which it is derived. In certain further embodiments of any of theabove, the Shiga toxin effector polypeptide is capable of exhibiting atleast one Shiga toxin effector function selected from: directingintracellular routing to a cytosol of a cell in which the polypeptide ispresent, inhibiting a ribosome function, enzymatically inactivating aribosome, and cytotoxicity.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fifthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the fifth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifth cell-targeting molecule toa second population of target positive cells as assayed by CD₅₀ values.

For certain embodiments of Embodiment Set #3, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #3, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., a sixthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

For certain further embodiments of Embodiment Set #3, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the fifth cell-targetingmolecule.

Embodiment Set #4—Cell-Targeting Molecule Comprising a Shiga ToxinEffector Polypeptide Comprising (i) an Embedded or Inserted,Heterologous, T-Cell Epitope and (ii) a Disrupted, Furin-Cleavage Motif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprisingan inserted or embedded, heterologous, epitope; and (iii) a disruptedfurin-cleavage motif. In certain embodiments, the cell-targetingmolecule of the present invention comprises (i) a binding region capableof specifically binding an extracellular target biomolecule; (ii) aShiga toxin effector polypeptide comprising (a) an inserted or embedded,heterologous, epitope; (b) a Shiga toxin A1 fragment derived regionhaving a carboxy terminus; and (c) a disrupted furin-cleavage motif atthe carboxy-terminus of the A1 fragment region. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface. For certainfurther embodiments, the cell-targeting molecule is capable whenintroduced to cells of exhibiting a cytotoxicity comparable or betterthan a reference molecule, such as, e.g., a second cell-targetingmolecule consisting of the cell-targeting molecule except for all of itsShiga toxin effector polypeptide components comprise a wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

In certain embodiments of Embodiment Set #4, the embedded or inserted,heterologous, T-cell epitope disrupts the endogenous, B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: (i) 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or theequivalent region in a Shiga toxin A Subunit or derivative thereof, (ii)94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ IDNO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 orSEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO: 3; 205 of SEQ IDNO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 ofSEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ IDNO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and285-293 of SEQ ID NO:1 or SEQ ID NO:2, or the equivalent region in aShiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Set #4, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ IDNO:2), or at 247-250 of the A Subunit of Shiga-like toxin (SEQ ID NO:3);or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, the disrupted furin-cleavagemotif comprises one or more mutations, relative to a wild-type Shigatoxin A Subunit, in a minimal furin cleavage site of the furin-cleavagemotif. In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #4, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #4, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #4, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #4, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a seventh cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment and/or wild-type Shiga toxin furin-cleavage site atthe carboxy terminus of its A1 fragment region. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable when introduced to cells of exhibiting cytotoxicity that is in arange of from 0.1-fold, 0.5-fold, or 0.75-fold to 1.2-fold, 1.5-fold,1.75-fold, 2-fold, 3-fold, 4-fold, or 5-fold of the cytotoxicityexhibited by the seventh cell-targeting molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of the seventhcell-targeting molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., an eighthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a ninth cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment.

Embodiment Set #5—Cell-Targeting Molecule Comprising a Shiga ToxinEffector Polypeptide at or Proximal to an Amino-Terminus and Wherein theShiga Toxin Effector Polypeptide Comprises an Embedded or Inserted,Heterologous, T-Cell Epitope

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprisingan inserted or embedded, heterologous, epitope; wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of apolypeptide. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule, (ii) a polypeptidecomponent, and (iii) a Shiga toxin effector polypeptide comprising aninserted or embedded, heterologous, epitope; wherein the Shiga toxineffector polypeptide is at or proximal to an amino-terminus of thepolypeptide component of the cell-targeting molecule. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Incertain further embodiments, the binding region is not locatedproximally to an amino-terminus of the cell-targeting molecule relativeto the Shiga toxin effector polypeptide. For certain furtherembodiments, the Shiga toxin effector polypeptide is capable ofexhibiting at least one Shiga toxin effector function, such as, e.g.,directing intracellular routing to the endoplasmic reticulum and/orcytosol of a cell in which the polypeptide is present, inhibiting aribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the heterologous, T-cell epitope is a CD8+ T-cell epitope, such as,e.g., with regard to a human immune system. For certain furtherembodiments, the heterologous, T-cell epitope is capable of beingpresented by a MHC class I molecule of a cell. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of one or more the following: entering a cell, inhibiting aribosome function, causing cytostasis, causing cell death, and/ordelivering the embedded or inserted, heterologous, T-cell epitope to aMHC class I molecule for presentation on a cellular surface.

In certain embodiments of Embodiment Set #5, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a tenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the tenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the tenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the tenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain embodiments of Embodiment Set #5, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Set #5, the cell-targeting moleculeis de-immunized due to the embedded or inserted, heterologous, epitope.In certain further embodiments, the cell-targeting molecule is capableof exhibiting less relative antigenicity and/or relative immunogenicityas compared to a reference molecule, such as, e.g., an eleventhcell-targeting molecule consisting of the cell-targeting molecule exceptfor it lacks one or more embedded or inserted epitopes present in thecell targeting molecule.

For certain further embodiments of Embodiment Set #5, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the tenth cell-targetingmolecule.

Embodiment Set #6—Cell-Targeting Molecule Comprising a De-ImmunizedShiga Toxin Effector Polypeptide Comprising a Disrupted, Furin-CleavageMotif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a de-immunized, Shiga toxin effectorpolypeptide comprising a disrupted furin-cleavage motif. In certainembodiments, the cell-targeting molecule of the present inventioncomprises (i) a binding region capable of specifically binding anextracellular target biomolecule and (ii) a de-immunized, Shiga toxineffector polypeptide comprising (a) a Shiga toxin A1 fragment derivedregion having a carboxy terminus, (b) a disrupted furin-cleavage motifat the carboxy-terminus of the A1 fragment region, and (c) at least onedisrupted, endogenous, B-cell and/or CD4+ T-cell epitope and/or epitoperegion. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath. For certain further embodiments, the cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor better than a reference molecule, such as, e.g., a secondcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide components comprise awild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #6, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218 of SEQ ID NO:3;240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ IDNO:3; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO:1 or SEQID NO:2; 34-78 of SEQ ID NO:1 or SEQ ID NO:2; 77-103 of SEQ ID NO:1 orSEQ ID NO:2; 128-168 of SEQ ID NO:1 or SEQ ID NO:2; 160-183 of SEQ IDNO:1 or SEQ ID NO:2; 236-258 of SEQ ID NO:1 or SEQ ID NO:2; and 274-293of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxinA Subunit or derivative thereof. In certain further embodiments, thereis no disruption which is a carboxy-terminal truncation of amino acidresidues that overlap with part or all of at least one disrupted,endogenous, B-cell and/or T-cell epitope and/or epitope region.

In certain embodiments of Embodiment Set #6, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ IDNO:2), or at 247-250 of the A Subunit of Shiga-like toxin (SEQ ID NO:3);or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, the disrupted furin-cleavagemotif comprises one or more mutations, relative to a wild-type Shigatoxin A Subunit, in a minimal furin cleavage site of the furin-cleavagemotif. In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #6, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #6, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #6, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #6, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #6, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #6, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #6, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #6, the cell-targeting moleculeis capable when introduced to cells of exhibiting cytotoxicitycomparable to the cytotoxicity of a reference molecule, such as, e.g., atwelfth cell-targeting molecule consisting of the cell-targetingmolecule except for all of its Shiga toxin effector polypeptidecomponent(s) each comprise a wild-type Shiga toxin A1 fragment and/orwild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region. In certain further embodiments, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is in a range of from 0.1-fold, 0.5-fold,or 0.75-fold to 1.2-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold,or 5-fold of the cytotoxicity exhibited by the twelfth cell-targetingmolecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of the twelfthcell-targeting molecule.

In certain embodiments of Embodiment Set #4, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the twelfthcell-targeting molecule.

Embodiment Set #7—Cell-Targeting Molecule Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and aDe-Immunized Shiga Toxin Effector Polypeptide

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a de-immunized, Shiga toxin effectorpolypeptide, and (iii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif. In certain embodiments, thecell-targeting molecule of the present invention comprises (i) a bindingregion capable of specifically binding an extracellular targetbiomolecule; (ii) a de-immunized, Shiga toxin effector polypeptidecomprising at least one disrupted, endogenous, B-cell and/or CD4+ T-cellepitope and/or epitope region, and (iii) a carboxy-terminal, endoplasmicreticulum retention/retrieval signal motif of a member of the KDELfamily. For certain further embodiments, the Shiga toxin effectorpolypeptide is capable of exhibiting at least one Shiga toxin effectorfunction, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath.

In certain embodiments of Embodiment Set #7, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL (SEQ ID NO: 514), HDEF (SEQ ID NO: 561),HDEL (SEQ ID NO: 515), RDEF (SEQ ID NO: 562), RDEL (SEQ ID NO: 516),WDEL (SEQ ID NO: 517), YDEL (SEQ ID NO: 518), HEEF (SEQ ID NO: 563),HEEL (SEQ ID NO: 519), KEEL (SEQ ID NO: 520), REEL (SEQ ID NO: 521),KAEL (SEQ ID NO: 564), KCEL (SEQ ID NO: 565), KFEL (SEQ ID NO: 522),KGEL (SEQ ID NO: 566), KHEL (SEQ ID NO: 567), KLEL (SEQ ID NO: 568),KNEL (SEQ ID NO: 569), KQEL (SEQ ID NO: 570), KREL (SEQ ID NO: 571),KSEL (SEQ ID NO: 572), KVEL (SEQ ID NO: 573), KWEL (SEQ ID NO: 574),KYEL (SEQ ID NO: 575), KEDL (SEQ ID NO: 576), KIEL (SEQ ID NO: 523),DKEL (SEQ ID NO: 524), FDEL (SEQ ID NO: 577), KDEF (SEQ ID NO: 578),KKEL (SEQ ID NO: 525), HADL (SEQ ID NO: 579), HAEL (SEQ ID NO: 580),HIEL (SEQ ID NO: 581), HNEL (SEQ ID NO: 526), HTEL (SEQ ID NO: 527),KTEL (SEQ ID NO: 528), HVEL (SEQ ID NO: 529), NDEL (SEQ ID NO: 582),QDEL (SEQ ID NO: 583), REDL (SEQ ID NO: 584), RNEL (SEQ ID NO: 585),RTDL (SEQ ID NO: 586), RTEL (SEQ ID NO: 587), SDEL (SEQ ID NO: 588),TDEL (SEQ ID NO: 589), SKEL (SEQ ID NO: 590), STEL (SEQ ID NO: 591), andEDEL (SEQ ID NO: 592).

In certain embodiments of Embodiment Set #7, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO: 2, and 210-218 of SEQ ID NO:3;240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ IDNO:3; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO:1 or SEQID NO:2; 34-78 of SEQ ID NO:1 or SEQ ID NO:2; 77-103 of SEQ ID NO:1 orSEQ ID NO:2; 128-168 of SEQ ID NO:1 or SEQ ID NO:2; 160-183 of SEQ IDNO:1 or SEQ ID NO:2; 236-258 of SEQ ID NO:1 or SEQ ID NO:2; and 274-293of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxinA Subunit or derivative thereof. In certain further embodiments, thereis no disruption which is a carboxy-terminal truncation of amino acidresidues that overlap with part or all of at least one disrupted,endogenous, B-cell and/or T-cell epitope and/or epitope region.

In certain embodiments of Embodiment Set #7, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a thirteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the thirteenth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the thirteenth cell-targetingmolecule to a second population of target positive cells as assayed byCD₅₀ values.

For certain further embodiments of Embodiment Set #7, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the thirteenth cell-targetingmolecule.

Embodiment Set #8—Cell-Targeting Molecule Comprising a De-ImmunizedShiga Toxin Effector Polypeptide at or Proximal to an Amino-Terminus ofthe Cell Targeting Molecule

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule, (ii) a de-immunized, Shiga toxin effectorpolypeptide; wherein the Shiga toxin effector polypeptide is at orproximal to an amino-terminus. In certain embodiments, thecell-targeting molecule of the present invention comprises (i) a bindingregion capable of specifically binding an extracellular targetbiomolecule; (ii) polypeptide component; and (iii) a de-immunized, Shigatoxin effector polypeptide comprising at least one disrupted,endogenous, B-cell and/or CD4+ T-cell epitope and/or epitope region;wherein the Shiga toxin effector polypeptide is at or proximal to anamino-terminus of the polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region and Shigatoxin effector polypeptide are physically arranged or oriented withinthe cell-targeting molecule such that the binding region is not locatedproximally to the amino-terminus of the Shiga toxin effectorpolypeptide. In certain further embodiments, the binding region islocated within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. For certain further embodiments, the Shigatoxin effector polypeptide is capable of exhibiting at least one Shigatoxin effector function, such as, e.g., directing intracellular routingto the endoplasmic reticulum and/or cytosol of a cell in which thepolypeptide is present, inhibiting a ribosome function, enzymaticallyinactivating a ribosome, causing cytostasis, and/or causingcytotoxicity. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of one or more thefollowing: entering a cell, inhibiting a ribosome function, causingcytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #8, the Shiga toxin effectorpolypeptide comprises a mutation, relative to a wild-type Shiga toxin ASubunit, in the B-cell and/or T-cell epitope region selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 ofSEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 94-115 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2, and 210-218 of SEQ ID NO:3;240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ IDNO:3; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 4-33 of SEQ ID NO:1 or SEQID NO:2; 34-78 of SEQ ID NO:1 or SEQ ID NO:2; 77-103 of SEQ ID NO:1 orSEQ ID NO:2; 128-168 of SEQ ID NO:1 or SEQ ID NO:2; 160-183 of SEQ IDNO:1 or SEQ ID NO:2; 236-258 of SEQ ID NO:1 or SEQ ID NO:2; and 274-293of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxinA Subunit or derivative thereof. In certain further embodiments, thereis no disruption which is a carboxy-terminal truncation of amino acidresidues that overlap with part or all of at least one disrupted,endogenous, B-cell and/or T-cell epitope and/or epitope region.

In certain embodiments of Embodiment Set #8, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fourteenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the fourteenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the fourteenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the fourteenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain further embodiments of Embodiment Set #8, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the fourteenth cell-targetingmolecule.

Embodiment Set #9—Cell-Targeting Molecule Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and a Shiga ToxinEffector Polypeptide Comprising a Disrupted, Furin-Cleavage Motif

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprising adisrupted furin-cleavage motif, and (iii) a carboxy-terminal endoplasmicreticulum retention/retrieval signal motif.

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule; (ii) a Shiga toxin effector polypeptide comprising adisrupted furin-cleavage motif; and (iii) a carboxy-terminal,endoplasmic reticulum retention/retrieval signal motif of a member ofthe KDEL family. For certain further embodiments, the Shiga toxineffector polypeptide is capable of exhibiting at least one Shiga toxineffector function, such as, e.g., directing intracellular routing to theendoplasmic reticulum and/or cytosol of a cell in which the polypeptideis present, inhibiting a ribosome function, enzymatically inactivating aribosome, causing cytostasis, and/or causing cytotoxicity. In certainfurther embodiments, the cell-targeting molecule of the presentinvention is capable of one or more the following: entering a cell,inhibiting a ribosome function, causing cytostasis, and/or causing celldeath. For certain further embodiments, the cell-targeting molecule iscapable when introduced to cells of exhibiting a cytotoxicity comparableor better than a reference molecule, such as, e.g., a secondcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide components comprise awild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #9, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ IDNO:2), or at 247-250 of the A Subunit of Shiga-like toxin (SEQ ID NO:3);or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, the disrupted furin-cleavagemotif comprises one or more mutations, relative to a wild-type Shigatoxin A Subunit, in a minimal furin cleavage site of the furin-cleavagemotif. In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculecomprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #9, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #9, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeof the present invention comprises a binding region and/or molecularmoiety located carboxy-terminal to the carboxy-terminus of the Shigatoxin A1 fragment region. In certain further embodiments, the mass ofthe binding region and/or molecular moiety is at least 4.5 kDa, 6, kDa,9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa,100 kDa, or greater.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculecomprises a binding region with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein(e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #9, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #9, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeof the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a fifteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the cell-targeting molecule of the present invention iscapable of exhibiting a cytotoxicity with better optimized, cytotoxicpotency, such as, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greatercytotoxicity as compared to the fifteenth cell-targeting molecule. Incertain further embodiments, the cytotoxicity of the cell-targetingmolecule of the present invention to a population of target positivecells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor greater than the cytotoxicity of the fifteenth cell-targetingmolecule to a second population of target positive cells as assayed byCD₅₀ values.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeis capable when introduced to a chordate of exhibiting improved, in vivotolerability compared to in vivo tolerability of a sixteenthcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion.

In certain embodiments of Embodiment Set #9, the cell-targeting moleculeis de-immunized due to the furin-cleavage motif disruption. In certainfurther embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the sixteenthcell-targeting molecule.

For certain further embodiments of Embodiment Set #9, the cell-targetingmolecule of the present invention is not cytotoxic and is capable whenintroduced to cells of exhibiting a greater subcellular routingefficiency from an extracellular space to a subcellular compartment ofan endoplasmic reticulum and/or cytosol as compared to the cytotoxicityof a reference molecule, such as, e.g., the fifteenth cell-targetingmolecule.

Embodiment Set #10—Cell-Targeting Molecule Comprising a Furin-CleavageResistant Shiga Toxin Effector Polypeptide at or Proximal to anAmino-Terminus of the Cell Targeting Molecule

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule and (ii) a Shiga toxin effector polypeptidecomprising a disrupted furin-cleavage motif at the carboxy-terminus ofits Shiga toxin A1 fragment region; wherein the amino-terminus of theShiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain embodiments, the cell-targeting molecule of thepresent invention comprises (i) a binding region capable of specificallybinding an extracellular target biomolecule, (ii) a Shiga toxin effectorpolypeptide having an amino-terminus and a Shiga toxin A1 fragmentderived region having a carboxy terminus, and (iii) a disruptedfurin-cleavage motif at the carboxy-terminus of the A1 fragment region;wherein the binding region is not located proximally to theamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. In certain further embodiments, the bindingregion and Shiga toxin effector polypeptide are physically arranged ororiented within the cell-targeting molecule such that the binding regionis not located proximally to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the binding regionis located within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide. For certain further embodiments, the Shigatoxin effector polypeptide is capable of exhibiting at least one Shigatoxin effector function, such as, e.g., directing intracellular routingto the endoplasmic reticulum and/or cytosol of a cell in which thepolypeptide is present, inhibiting a ribosome function, enzymaticallyinactivating a ribosome, causing cytostasis, and/or causingcytotoxicity. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of one or more thefollowing: entering a cell, inhibiting a ribosome function, causingcytostasis, and/or causing cell death. For certain further embodiments,the cell-targeting molecule is capable when introduced to cells ofexhibiting a cytotoxicity comparable or better than a referencemolecule, such as, e.g., a seventeenth cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide components comprise a wild-type Shiga toxinfurin-cleavage site at the carboxy terminus of its A1 fragment region.

In certain embodiments of Embodiment Set #10, the disruptedfurin-cleavage motif comprises one or more mutations, relative to awild-type Shiga toxin A Subunit, the mutation altering at least oneamino acid residue in a region natively positioned at 248-251 of the ASubunit of Shiga-like toxin 1 (SEQ ID NO: 1) or Shiga toxin (SEQ IDNO:2), or at 247-250 of the A Subunit of Shiga-like toxin (SEQ ID NO:3);or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, the disrupted furin-cleavagemotif comprises one or more mutations, relative to a wild-type Shigatoxin A Subunit, in a minimal furin cleavage site of the furin-cleavagemotif. In certain further embodiments the minimal furin cleavage site isrepresented by the consensus amino acid sequence R/Y-x-x-R and/orR-x-x-R.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Set #10, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Set #10, the molecular moietysterically covers the carboxy-terminus of the A1 fragment region. Incertain further embodiments, the molecular moiety comprises the bindingregion.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater, as long as the cell-targeting molecule retainsthe appropriate level of the Shiga toxin biological activity notedherein (e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Set #10, the binding region iscomprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

In certain embodiments of Embodiment Set #10, the disruptedfurin-cleavage motif comprises an amino acid residue substitution in thefurin-cleavage motif relative to a wild-type Shiga toxin A Subunit. Incertain further embodiments, the substitution of the amino acid residuein the furin-cleavage motif is of an arginine residue with anon-positively charged, amino acid residue selected from the groupconsisting of: alanine, glycine, proline, serine, threonine, aspartate,asparagine, glutamate, glutamine, cysteine, isoleucine, leucine,methionine, valine, phenylalanine, tryptophan, and tyrosine. In certainembodiments, the substitution of the amino acid residue in thefurin-cleavage motif is of an arginine residue with a histidine.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a eighteenthcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the eighteenthcell-targeting molecule. In certain further embodiments, thecell-targeting molecule of the present invention is capable ofexhibiting a cytotoxicity with better optimized, cytotoxic potency, suchas, e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the eighteenth cell-targeting molecule. In certain furtherembodiments, the cytotoxicity of the cell-targeting molecule of thepresent invention to a population of target positive cells is 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or greater thanthe cytotoxicity of the eighteenth cell-targeting molecule to a secondpopulation of target positive cells as assayed by CD₅₀ values.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule is capable when introduced to a chordate of exhibitingimproved, in vivo tolerability compared to in vivo tolerability of anineteenth cell-targeting molecule consisting of the cell-targetingmolecule except for all of its Shiga toxin effector polypeptidecomponent(s) each comprise a wild-type Shiga toxin A1 fragment and/orwild-type Shiga toxin furin-cleavage site at the carboxy terminus of itsA1 fragment region.

In certain embodiments of Embodiment Set #10, the cell-targetingmolecule is de-immunized due to the furin-cleavage motif disruption. Incertain further embodiments, the cell-targeting molecule is capable ofexhibiting less relative antigenicity and/or relative immunogenicity ascompared to a reference cell-targeting molecule consisting of thecell-targeting molecule except for the furin-cleavage motif is wild-typeand/or all the Shiga toxin effector polypeptide components consist of awild-type Shiga toxin A1 fragment, such as, e.g., the nineteenthcell-targeting molecule.

For certain further embodiments of Embodiment Set #10, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the nineteenthcell-targeting molecule.

Embodiment Set #11—Cell-Targeting Molecule Comprising a Carboxy-TerminalEndoplasmic Reticulum Retention/Retrieval Signal Motif and Shiga ToxinEffector Polypeptide at or Proximal to an Amino-Terminus of the CellTargeting Molecule

The present invention provides cell-targeting molecules, each comprising(i) a binding region capable of specifically binding an extracellulartarget biomolecule, (ii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif, and (iii) a Shiga toxin effectorpolypeptide; wherein the amino-terminus of the Shiga toxin effectorpolypeptide is at and/or proximal to an amino-terminus of a polypeptidecomponent of the cell-targeting molecule. In certain embodiments, thecell-targeting molecule of the present invention comprises a (i) bindingregion capable of specifically binding an extracellular targetbiomolecule, (ii) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of a member of the KDEL family, (iii) apolypeptide component, and (iv) a Shiga toxin effector polypeptide;wherein the amino-terminus of the Shiga toxin effector polypeptide is atand/or proximal to an amino-terminus of a polypeptide component of thecell-targeting molecule. In certain further embodiments, the bindingregion and Shiga toxin effector polypeptide are physically arranged ororiented within the cell-targeting molecule such that the binding regionis not located proximally to the amino-terminus of the Shiga toxineffector polypeptide. In certain further embodiments, the binding regionis located within the cell-targeting molecule more proximal to thecarboxy-terminus of the Shiga toxin effector polypeptide than to theamino-terminus of the Shiga toxin effector polypeptide. In certainfurther embodiments, the binding region is not located proximally to anamino-terminus of the cell-targeting molecule relative to the Shigatoxin effector polypeptide.

For certain further embodiments, the Shiga toxin effector polypeptide iscapable of exhibiting at least one Shiga toxin effector function, suchas, e.g., directing intracellular routing to the endoplasmic reticulumand/or cytosol of a cell in which the polypeptide is present, inhibitinga ribosome function, enzymatically inactivating a ribosome, causingcytostasis, and/or causing cytotoxicity. In certain further embodiments,the cell-targeting molecule of the present invention is capable of oneor more the following: entering a cell, inhibiting a ribosome function,causing cytostasis, and/or causing cell death.

In certain embodiments of Embodiment Set #11, the carboxy-terminalendoplasmic reticulum retention/retrieval signal motif is selected fromthe group consisting of: KDEL (SEQ ID NO: 514), HDEF (SEQ ID NO: 561),HDEL (SEQ ID NO: 515), RDEF (SEQ ID NO: 562), RDEL (SEQ ID NO: 516),WDEL (SEQ ID NO: 517), YDEL (SEQ ID NO: 518), HEEF (SEQ ID NO: 563),HEEL (SEQ ID NO: 519), KEEL (SEQ ID NO: 520), REEL (SEQ ID NO: 521),KAEL (SEQ ID NO: 564), KCEL (SEQ ID NO: 565), KFEL (SEQ ID NO: 522),KGEL (SEQ ID NO: 566), KHEL (SEQ ID NO: 567), KLEL (SEQ ID NO: 568),KNEL (SEQ ID NO: 569), KQEL (SEQ ID NO: 570), KREL (SEQ ID NO: 571),KSEL (SEQ ID NO: 572), KVEL (SEQ ID NO: 573), KWEL (SEQ ID NO: 574),KYEL (SEQ ID NO: 575), KEDL (SEQ ID NO: 576), KIEL (SEQ ID NO: 523),DKEL (SEQ ID NO: 524), FDEL (SEQ ID NO: 577), KDEF (SEQ ID NO: 578),KKEL (SEQ ID NO: 525), HADL (SEQ ID NO: 579), HAEL (SEQ ID NO: 580),HIEL (SEQ ID NO: 581), HNEL (SEQ ID NO: 526), HTEL (SEQ ID NO: 527),KTEL (SEQ ID NO: 528), HVEL (SEQ ID NO: 529), NDEL (SEQ ID NO: 582),QDEL (SEQ ID NO: 583), REDL (SEQ ID NO: 584), RNEL (SEQ ID NO: 585),RTDL (SEQ ID NO: 586), RTEL (SEQ ID NO: 587), SDEL (SEQ ID NO: 588),TDEL (SEQ ID NO: 589), SKEL (SEQ ID NO: 590), STEL (SEQ ID NO: 591), andEDEL (SEQ ID NO: 592).

In certain embodiments of Embodiment Set #11, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity that is greater than that of a twentiethcell-targeting molecule having an amino-terminus and comprising thebinding region and the Shiga toxin effector polypeptide region which isnot positioned at or proximal to the amino-terminus of the twentiethcell-targeting molecule and/or greater than that of a twenty-firstcell-targeting molecule consisting of the cell-targeting molecule exceptfor it does not comprise any carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif of the KDEL family. In certain furtherembodiments, the twentieth cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the twentieth and/or twenty-firstcell-targeting molecules. In certain further embodiments, thecytotoxicity of the cell-targeting molecule of the present invention toa population of target positive cells is 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold or greater than the cytotoxicity of thetwentieth and/or twenty-first cell-targeting molecules to a secondpopulation of target positive cells as assayed by CD₅₀ values.

For certain further embodiments of Embodiment Set #11, thecell-targeting molecule of the present invention is not cytotoxic and iscapable when introduced to cells of exhibiting a greater subcellularrouting efficiency from an extracellular space to a subcellularcompartment of an endoplasmic reticulum and/or cytosol as compared tothe cytotoxicity of a reference molecule, such as, e.g., the twentiethand/or twenty-first cell-targeting molecules.

Further Embodiments of Embodiment Sets #1-#11

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide is fused to the binding region, either directly orindirectly, such as, e.g., via a linker known to the skilled worker.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule comprises a molecular moiety located carboxy-terminal to thecarboxy-terminus of the Shiga toxin A1 fragment region.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide has a Shiga toxin A1 fragment derived region havinga carboxy terminus and further comprises a disrupted furin-cleavagemotif at the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention, or a polypeptide component thereof,comprises a carboxy-terminal, endoplasmic reticulum retention/retrievalsignal motif of a member of the KDEL family. For certain furtherembodiments, the carboxy-terminal endoplasmic reticulumretention/retrieval signal motif is selected from the group consistingof: KDEL (SEQ ID NO: 514), HDEF (SEQ ID NO: 561), HDEL (SEQ ID NO: 515),RDEF (SEQ ID NO: 562), RDEL (SEQ ID NO: 516), WDEL (SEQ ID NO: 517),YDEL (SEQ ID NO: 518), HEEF (SEQ ID NO: 563), HEEL (SEQ ID NO: 519),KEEL (SEQ ID NO: 520), REEL (SEQ ID NO: 521), KAEL (SEQ ID NO: 564),KCEL (SEQ ID NO: 565), KFEL (SEQ ID NO: 522), KGEL (SEQ ID NO: 566),KHEL (SEQ ID NO: 567), KLEL (SEQ ID NO: 568), KNEL (SEQ ID NO: 569),KQEL (SEQ ID NO: 571), KREL (SEQ ID NO: 571), KSEL (SEQ ID NO: 572),KVEL (SEQ ID NO: 573), KWEL (SEQ ID NO: 574), KYEL (SEQ ID NO: 575),KEDL (SEQ ID NO: 576), KIEL (SEQ ID NO: 523), DKEL (SEQ ID NO: 524),FDEL (SEQ ID NO: 577), KDEF (SEQ ID NO: 578), KKEL (SEQ ID NO: 525),HADL (SEQ ID NO: 579), HAEL (SEQ ID NO: 580), HIEL (SEQ ID NO: 581),HNEL (SEQ ID NO: 526), HTEL (SEQ ID NO: 527), KTEL (SEQ ID NO: 528),HVEL (SEQ ID NO: 529), NDEL (SEQ ID NO: 582), QDEL (SEQ ID NO: 583),REDL (SEQ ID NO: 584), RNEL (SEQ ID NO: 585), RTDL (SEQ ID NO: 586),RTEL (SEQ ID NO: 587), SDEL (SEQ ID NO: 588), TDEL (SEQ ID NO: 589),SKEL (SEQ ID NO: 590), STEL (SEQ ID NO: 591), and EDEL (SEQ ID NO: 592).In certain further embodiments, the cell-targeting molecule of thepresent invention is capable when introduced to cells of exhibitingcytotoxicity that is greater than that of a reference molecule, such as,e.g., a twenty-second cell-targeting molecule consisting of thecell-targeting molecule except for it does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family. In certain further embodiments, the cell-targetingmolecule of the present invention is capable of exhibiting acytotoxicity with better optimized, cytotoxic potency, such as, e.g.,4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity as compared to areference molecule, such as, e.g., the twenty-second cell-targetingmolecule. In certain further embodiments, the cytotoxicity of thecell-targeting molecule of the present invention to a population oftarget positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold or greater than the cytotoxicity of the twenty-secondcell-targeting molecule to a second population of target positive cellsas assayed by CD₅₀ values.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide further comprises at least one inserted orembedded, heterologous epitope.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide further comprises at least one, two, or threedisrupted, endogenous, B-cell and/or CD4+ T-cell epitope regions. Incertain further embodiments, the Shiga toxin effector polypeptidecomprises a disruption of at least one, two, or three endogenous, B-celland/or T-cell epitopes and/or epitope regions. In certain furtherembodiments, the Shiga toxin effector polypeptide further comprises atleast one disrupted, endogenous, B-cell and/or CD4+ T-cell epitoperegion which does not overlap with at least one inserted or embedded,heterologous epitope.

In certain embodiments of Embodiment Sets #2 to #11, the amino-terminusof the Shiga toxin effector polypeptide is at and/or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule. In certain further embodiments, the binding region is notlocated proximally to the amino-terminus of the cell-targeting moleculerelative to the Shiga toxin effector polypeptide. In certain furtherembodiments, the binding region and Shiga toxin effector polypeptide arephysically arranged or oriented within the cell-targeting molecule suchthat the binding region is not located proximally to the amino-terminusof the Shiga toxin effector polypeptide. In certain further embodiments,the binding region is located within the cell-targeting molecule moreproximal to the carboxy-terminus of the Shiga toxin effector polypeptidethan to the amino-terminus of the Shiga toxin effector polypeptide. Forcertain further embodiments, the cell-targeting molecule of the presentinvention is not cytotoxic and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., a twenty-third cell-targeting molecule havingan amino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. For certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits cytotoxicity with better optimized, cytotoxic potency, such as,e.g., 4-fold, 5-fold, 6-fold, 9-fold, or greater cytotoxicity ascompared to the cytotoxicity of the twenty-third cell-targetingmolecule. For certain further embodiments, the cytotoxicity of thecell-targeting molecule of the present invention to a population oftarget positive cells is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold or greater than the cytotoxicity of the twenty-thirdcell-targeting molecule to a second population of target positive cellsas assayed by CD₅₀ values. In certain further embodiments, thetwenty-third cell-targeting molecule does not comprise anycarboxy-terminal, endoplasmic reticulum retention/retrieval signal motifof the KDEL family.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide further comprises a disruption in the B-cell and/orT-cell epitope region selected from the group of natively positionedShiga toxin A Subunit regions consisting of: 1-15 of SEQ ID NO:1 or SEQID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO: 3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO: 3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQID NO:2, and 210-218 of SEQ ID NO: 3; 240-260 of SEQ ID NO:3; 243-257 ofSEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2;262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; 285-293 of SEQ ID NO:1or SEQ ID NO:2; 4-33 of SEQ ID NO:1 or SEQ ID NO:2; 34-78 of SEQ ID NO:1or SEQ ID NO:2; 77-103 of SEQ ID NO:1 or SEQ ID NO:2; 128-168 of SEQ IDNO:1 or SEQ ID NO:2; 160-183 of SEQ ID NO:1 or SEQ ID NO:2; 236-258 ofSEQ ID NO:1 or SEQ ID NO:2; and 274-293 of SEQ ID NO:1 or SEQ ID NO:2;or the equivalent region in a Shiga toxin A Subunit or derivativethereof. In certain further embodiments, there is no disruption which isa carboxy-terminal truncation of amino acid residues that overlap withpart or all of at least one disrupted, endogenous, B-cell and/or T-cellepitope and/or epitope region.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide further comprises a mutation, relative to awild-type Shiga toxin A Subunit, in the B-cell immunogenic, amino acidresidue selected from the group of natively positioned Shiga toxin ASubunit amino acid residues: L49, D197, D198, R204, and R205.

In certain embodiments of Embodiment Sets #2 to #11, the embedded orinserted, heterologous, T-cell epitope disrupts the endogenous, B-celland/or T-cell epitope region is selected from the group of nativelypositioned Shiga toxin A Subunit regions consisting of: (i) 1-15 of SEQID NO:1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37of SEQ ID NO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2;42-48 of SEQ ID NO:3; and 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; or the equivalent region in a Shiga toxin A Subunit or derivativethereof, wherein there is no disruption which is an amino-terminaltruncation of sequences that overlap with part or all of at least onedisrupted epitope region; (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ IDNO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO: 2; 179-191 of SEQ ID NO:3;204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 ofSEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 orSEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ IDNO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2;or the equivalent region in a Shiga toxin A Subunit or derivativethereof.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide comprises a mutation, relative to a wild-type Shigatoxin A Subunit, in the B-cell and/or T-cell epitope region selectedfrom the group of natively positioned Shiga toxin A Subunit regionsconsisting of: (i) 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ IDNO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; or the equivalent region in a Shigatoxin A Subunit or derivative thereof, wherein there is no disruptionwhich is an amino-terminal truncation of sequences that overlap withpart or all of at least one disrupted epitope region; (ii) 94-115 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ IDNO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; and 210-218 of SEQ ID NO:3; and (iii) 240-260 of SEQ ID NO:3;243-257 of SEQ ID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ IDNO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQID NO:1 or SEQ ID NO:2; or the equivalent region in a Shiga toxin ASubunit or derivative thereof, wherein there is no disruption which isan amino-terminal truncation of sequences that overlap with part or allof at least one disrupted epitope region.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide comprises a disruption of at least one endogenousepitope region selected from the group of natively positioned Shigatoxin A Subunits consisting of: 94-115 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ IDNO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; or 210-218 of SEQ IDNO:3.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide does not comprise a heterologous, MHC classI-restricted, T-cell epitope. MHC class I-restricted, T-cell epitopesare known in the art or can be predicted by the skilled worker. The termheterologous refers to MHC class I-restricted, T-cell epitopes which arenot natively present in wild-type Shiga toxin A Subunits, such as, e.g.,the wild-type Shiga toxin A Subunit which is most closely related to theShiga toxin effector polypeptide of interest.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide comprises disruptions of at least four, five, six,seven, eight, or more endogenous, B-cell and/or T-cell epitope regions.

In certain embodiments of Embodiment Sets #2 to #11, one or moredisruptions comprises an amino acid residue substitution relative to awild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #2 to #11, one or moreendogenous, B-cell and/or T-cell epitope regions comprises a pluralityof amino acid residue substitutions relative to a wild-type Shiga toxinA Subunit.

In certain embodiments of Embodiment Sets #2 to #11, at least one, two,three, or four disruptions comprise a plurality of amino acid residuesubstitutions in the endogenous, B-cell and/or T-cell epitope regionrelative to a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #2 to #11, at least onedisruption comprises at least one, two, three, four, five, six, seven,eight, or more amino acid residue substitutions relative to a wild-typeShiga toxin A Subunit, and optionally wherein at least one substitutionoccurs at the natively positioned Shiga toxin A Subunit amino acidresidue selected form the group consisting of: 1 of SEQ ID NO:1 or SEQID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 11 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQID NO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ IDNO:2; 46 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQ ID NO:1or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 50 of SEQ ID NO:1 or SEQ IDNO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ IDNO:2; 54 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQ ID NO:1or SEQ ID NO: 2; 56 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 57 ofSEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO:3; 58 of SEQ ID NO: 1, SEQ IDNO:2, or SEQ ID NO: 3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3;60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3;104 of SEQ ID NO:1 or SEQ ID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2;107 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 orSEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;112 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO: 1,SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO:1, SEQ ID NO: 2, or SEQ IDNO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ IDNO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 orSEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ IDNO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ IDNO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO: 3; 248 of SEQ ID NO:1 or SEQID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ IDNO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2; or the equivalent aminoacid residue in a Shiga toxin A Subunit or derivative thereof. Incertain further embodiments, at least two disruptions each comprise atleast one amino acid residue substitutions relative to a wild-type Shigatoxin A Subunit selected form the group consisting of: 1 of SEQ ID NO:1or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ IDNO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 53of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 58 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO: 2; 61 of SEQ IDNO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 94 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO: 1, SEQ ID NO:2, orSEQ ID NO: 3; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 ofSEQ ID NO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 179 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2;181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID SEQ IDNO:2, or SEQ ID NO:3; 184 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3;185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 orSEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 ofSEQ ID NO: 3; 250 of SEQ ID NO:3; 264 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1or SEQ ID NO:2; or the equivalent amino acid residue in a Shiga toxin ASubunit or derivative thereof.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide comprises disruption of at least three, endogenous,B-cell and/or T-cell epitope regions selected from the group ofconsisting of: (i) 1-15 of SEQ ID NO:1 or SEQ ID NO:2; 3-14 of SEQ IDNO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 or SEQ ID NO:2; 39-48of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; and 53-66 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3, or the equivalent region in a Shigatoxin A Subunit or derivative thereof, wherein there is no disruptionwhich is an amino-terminal truncation of amino acid residues thatoverlap with part or all of at least one disrupted, endogenous, B-celland/or T-cell epitope region; (ii) 94-115 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ IDNO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ IDNO: 3; and (iii) 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO:1 or SEQID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3;281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 or SEQ ID NO:2; orthe equivalent region in a Shiga toxin A Subunit or derivative thereof,wherein there is no disruption which is a carboxy-terminal truncation ofamino acid residues that overlap with part or all of at least onedisrupted, endogenous, B-cell and/or T-cell epitope and/or epitoperegion.

In certain embodiments of Embodiment Sets #2 to #11, the Shiga toxineffector polypeptide comprises disruptions of at least two, endogenous,B-cell and/or T-cell epitope regions, wherein each disruption comprisesone or more amino acid residue substitutions, and wherein theendogenous, B-cell and/or T-cell epitope regions are selected from thegroup of natively positioned Shiga toxin A Subunit regions consistingof: 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO:1 orSEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3;53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; or the equivalentregion in a Shiga toxin A Subunit or derivative thereof.

In certain embodiments of Embodiment Sets #2 to #11, the embedded orinserted, heterologous, T-cell epitope does not disrupt any endogenous,B-cell and/or CD4+ T-cell epitope region described herein.

In certain embodiments of Embodiment Sets #2 to #11, at least onedisruption comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, D to M, D to R, E to A, E to G, E to V, E to L, E to I, E to F, Eto S, E to Q, E to N, E to D, E to M, E to R, F to A, F to G, F to V, Fto L, F to I, G to A, G to P, H to A, H to G, H to V, H to L, H to I, Hto F, H to M, I to A, I to V, I to G, I to C, K to A, K to G, K to V, Kto L, K to I, K to M, K to H, L to A, L to V, L to G, L to C, N to A, Nto G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, Rto G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, Rto H, S to A, S to G, S to V, S to L, S to I, S to F, S to M, T to A, Tto G, T to V, T to L, T to I, T to F, T to M, T to S, V to A, V to G, Yto A, Y to G, Y to V, Y to L, Y to I, Y to F, Y to M, and Y to T. Incertain further embodiments, the one or more amino acid residuesubstitutions relative to a wild-type Shiga toxin A Subunit is selectedfrom the group consisting of: D to A, D to G, D to V, D to L, D to I, Dto F, D to S, D to Q, E to A, E to G, E to V, E to L, E to I, E to F, Eto S, E to Q, E to N, E to D, E to M, E to R, G to A, H to A, H to G, Hto V, H to L, H to I, H to F, H to M, K to A, K to G, K to V, K to L, Kto I, K to M, K to H, L to A, L to G, N to A, N to G, N to V, N to L, Nto I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, Rto I, R to F, R to M, R to Q, R to S, R to K, R to H, S to A, S to G, Sto V, S to L, S to I, S to F, S to M, T to A, T to G, T to V, T to L, Tto I, T to F, T to M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Yto F, and Y to M.

In certain embodiments of Embodiment Sets #2 to #11, at least one of thedisruption(s) comprises one or more amino acid residue substitutionsrelative to a wild-type Shiga toxin A Subunit selected from the groupconsisting of: K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F,M, and S; D6 to A, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F,and M; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M;K11 to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K;S12 to A, G, V, I, L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43to A, G, V, L, I, F, and M; G44 to A or L; S45 to A, G, V, L, I, F, andM; T45 to A, G, V, L, I, F, and M; G46 to A and P; D47 to A, G, V, L, I,F, S, M, and Q; N48 to A, G, V, L, M and F; L49 to A, V, C, and G; Y49to A, G, V, L, I, F, M, and T; F50 to A, G, V, L, I, and T; A51; D53toA, G, V, L, I, F, S, and Q; V54 to A, G, I, and L; R55to A, G, V, L, I,F, M, Q, S, K, and H; G56 to A and P; 157 to A, G, V, and M; L57 to A,V, C, G, M, and F; D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, andF; E60 to A, G, V, L, I, F, S, Q, N, D, M, T, and R; E61 to A, G, V, L,I, F, S, Q, N, D, M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S,K, and H; V88 to A and G; 188 to A, V, C, and G; D94 to A, G, V, L, I,F, S, and Q; S96 to A, G, V, I, L, F, and M; T104 to A, G, V, L, I, F,M; and N; A105 to L; T107 to A, G, V, L, I, F, M, and P; S107 to A, G,V, L, I, F, M, and P; L108 to A, V, C, and G; S109 to A, G, V, I, L, F,and M; T109 to A, G, V, I, L, F, M, and S; G110 to A; S112 to A, G, V,L, I, F, and M; D111 to A, G, V, L, I, F, S, Q, and T; S112 to A, G, V,L, I, F, and M; D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to Aand G. R179 to A, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L,I, F, M, and S; T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L,I, F, S, and Q; D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, V andC; S186 to A, G, V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F,M, Q, S, K, and H; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L,I, F, S, and Q; D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L,I, F, M, Q, S, K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H;S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R248to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q,S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G,V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S.

For certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention is capable when introduced to achordate of exhibiting improved in vivo tolerability and/or stabilitycompared to a reference molecule, such as, e.g., a twenty-fourthcell-targeting molecule consisting of the cell-targeting molecule exceptfor all of its Shiga toxin effector polypeptide component(s) eachcomprise a wild-type Shiga toxin A1 fragment and/or wild-type Shigatoxin furin-cleavage site at the carboxy terminus of its A1 fragmentregion. In certain further embodiments, the Shiga toxin effectorpolypeptide is not cytotoxic and the molecular moiety is cytotoxic.

In certain embodiments of Embodiment Sets #2 to #11, the binding regionand Shiga toxin effector polypeptide are linked together, eitherdirectly or indirectly.

In certain embodiments of Embodiment Sets #2 to #11, the binding regioncomprises at least one peptide and/or polypeptide. In certain furtherembodiments, the binding region is or comprises an immunoglobulin-typebinding region. In certain further embodiments, the binding regioncomprising a polypeptide selected from the group consisting of: anautonomous V_(H) domain, single-domain antibody fragment (sdAb),nanobody, heavy chain-antibody domain derived from a camelid (V_(H)H orV_(H) domain fragment), heavy-chain antibody domain derived from acartilaginous fish (V_(H)H or V_(H) domain fragment), immunoglobulin newantigen receptor (IgNAR), V_(NAR) fragment, single-chain variablefragment (scFv), antibody variable fragment (Fv), complementarydetermining region 3 fragment (CDR3), constrained FR3-CDR3-FR4polypeptide (FR3-CDR3-FR4), Fd fragment, small modularimmunopharmaceutical (SMIP) domain, antigen-binding fragment (Fab),Armadillo repeat polypeptide (ArmRP), fibronectin-derived 10^(th)fibronectin type III domain (10Fn3), tenascin type III domain (TNfn3),ankyrin repeat motif domain, low-density-lipoprotein-receptor-derivedA-domain (LDLR-A), lipocalin (anticalin), Kunitz domain,Protein-A-derived Z domain, gamma-B crystalline-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide (affitin),Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold,engineered antibody mimic, and any genetically manipulated counterpartsof any of the foregoing which retain binding functionality.

For certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention is capable of exhibiting (i) acatalytic activity level comparable to a wild-type Shiga toxin A1fragment or wild-type Shiga toxin effector polypeptide, (ii) a ribosomeinhibition activity with a half-maximal inhibitory concentration (IC₅₀)value of 10,000 picomolar or less, and/or (iii) a significant level ofShiga toxin catalytic activity.

For certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention and/or its Shiga toxin effectorpolypeptide is capable of exhibiting subcellular routing efficiencycomparable to a reference cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment or wild-type Shiga toxin effector polypeptideand/or capable of exhibiting a significant level of intracellularrouting activity to the endoplasmic reticulum and/or cytosol from anendosomal starting location of a cell.

For certain embodiments of Embodiment Sets #2 to #11, wherebyadministration of the cell-targeting molecule of the present inventionto a cell physically coupled with the extracellular target biomoleculeof the cell-targeting molecule's binding region, the cell-targetingmolecule is capable of causing death of the cell. In certain furtherembodiments, administration of the cell-targeting molecule of theinvention to two different populations of cell types which differ withrespect to the presence or level of the extracellular targetbiomolecule, the cell-targeting molecule is capable of causing celldeath to the cell-types physically coupled with an extracellular targetbiomolecule of the cytotoxic cell-targeting molecule's binding region ata CD₅₀ at least three times or less than the CD₅₀ to cell types whichare not physically coupled with an extracellular target biomolecule ofthe cell-targeting molecule's binding region. For certain embodiments,whereby administration of the cell-targeting molecule of the presentinvention to a first populations of cells whose members are physicallycoupled to extracellular target biomolecules of the cell-targetingmolecule's binding region, and a second population of cells whosemembers are not physically coupled to any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationsof cells whose members are physically coupled to a significant amount ofthe extracellular target biomolecule of the cell-targeting molecule'sbinding region, and a second population of cells whose members are notphysically coupled to a significant amount of any extracellular targetbiomolecule of the binding region, the cytotoxic effect of thecell-targeting molecule to members of said first population of cellsrelative to members of said second population of cells is at least3-fold greater. For certain embodiments, whereby administration of thecell-targeting molecule of the present invention to a first populationof target biomolecule positive cells, and a second population of cellswhose members do not express a significant amount of a targetbiomolecule of the cell-targeting molecule's binding region at acellular surface, the cytotoxic effect of the cell-targeting molecule tomembers of the first population of cells relative to members of thesecond population of cells is at least 3-fold greater.

For certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting a cytotoxicity with a half-maximal inhibitory concentration(CD₅₀) value of 300 nM or less and/or capable of exhibiting asignificant level of Shiga toxin cytotoxicity.

For certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention is capable of delivering an embeddedor inserted, heterologous, CD8+ T-cell epitope to a MHC class Ipresentation pathway of a cell for cell-surface presentation of theepitope bound by a MHC class I molecule.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule comprises a molecular moiety associated with thecarboxy-terminus of the Shiga toxin effector polypeptide. In certainembodiments, the molecular moiety comprises or consists of the bindingregion. In certain embodiments, the molecular moiety comprises at leastone amino acid and the Shiga toxin effector polypeptide is linked to atleast one amino acid residue of the molecular moiety. In certain furtherembodiments, the molecular moiety and the Shiga toxin effectorpolypeptide are fused forming a continuous polypeptide.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule further comprises a cytotoxic molecular moiety associated withthe carboxy-terminus of the Shiga toxin effector polypeptide. Forcertain embodiments, the cytotoxic molecular moiety is a cytotoxicagent, such as, e.g., a small molecule chemotherapeutic agent,anti-neoplastic agent, cytotoxic antibiotic, alkylating agent,antimetabolite, topoisomerase inhibitor, and/or tubulin inhibitor knownto the skilled worker and/or described herein. For certain furtherembodiments, the cytotoxic molecular moiety is cytotoxic atconcentrations of less than 10,000, 5,000, 1,000, 500, or 200 pM.

In certain embodiments of Embodiment Sets #2 to #11, the binding regionis capable of binding to an extracellular target biomolecule selectedfrom the group consisting of: CD20, CD22, CD40, CD74, CD79, CD25, CD30,HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen,Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19,CD21, CS1/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72,tyrosine-protein kinase transmembrane receptor (ROR1 or NTRKR1),carbonic anhydrase IX, folate binding protein, ganglioside GD2,ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, AlphaVbeta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGF1R, EphA3, TRAIL-R1,TRAIL-R2, RANK, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA,gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE,NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6,SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen,prostate stem cell antigen, human aspartyl (asparaginyl)beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gp100,tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen,Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38,CD15, CD23, CD45 (protein tyrosine phosphatase receptor type C), CD53,CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa,galectin-9, IL-1R (interleukin-1 receptor), mrp-14, NKG2D ligand,programmed death-ligand 1 (PD-L1), Siglec-8, Siglec-10, CD49d, CD13,CD44, CD54, CD63, CD69, CD123, TLR4, FeeRIa, IgE, CD107a, CD203c, CD14,CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c,GITRL, MHC class I molecule, MHC class II molecule (optionally complexedwith a peptide), CD284 (TLR4), CD107-Mac3, CD195 (CCR5), HLA-DR,CD16/32, CD282 (TLR2), CD11c, and any immunogenic fragment of any of theforegoing.

In certain embodiments of Embodiment Sets #2 to #11, the binding regionis linked, either directly or indirectly, to the Shiga toxin effectorpolypeptide by at least one covalent bond which is not a disulfide bond.In certain further embodiments, the binding region is fused, eitherdirectly or indirectly, to the carboxy-terminus of the Shiga toxineffector polypeptide to form a single, continuous polypeptide. Incertain further embodiments, the binding region is animmunoglobulin-type binding region.

In certain embodiments of Embodiment Sets #2 to #11, the disruptedfurin-cleavage motif comprises one or more mutations in the minimal,furin-cleavage site relative to a wild-type Shiga toxin A Subunit. Incertain embodiments, the disrupted furin-cleavage motif is not anamino-terminal truncation of sequences that overlap with part or all ofat least one amino acid residue of the minimal furin-cleavage site. Incertain embodiments, the mutation in the minimal, furin-cleavage site isan amino acid deletion, insertion, and/or substitution of at least oneamino acid residue in the R/Y-x-x-R furin cleavage motif. In certainfurther embodiments, the disrupted furin-cleavage motif comprises atleast one mutation relative to a wild-type Shiga toxin A Subunit, themutation altering at least one amino acid residue in the region nativelypositioned 1) at 248-251 of the A Subunit of Shiga-like toxin 1 (SEQ IDNO: 1) or Shiga toxin (SEQ ID NO: 2), or 2) at 247-250 of the A Subunitof Shiga-like toxin 2 (SEQ ID NO:3), or the equivalent amino acidsequence position in any Shiga toxin A Subunit. In certain furtherembodiments, the mutation is an amino acid residue substitution of anarginine residue with a non-positively charged, amino acid residue.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention is capable when introduced to cells ofexhibiting cytotoxicity comparable to a cytotoxicity of a referencemolecule, such as, e.g., a twenty-fifth cell-targeting moleculeconsisting of the cell-targeting molecule except for all of its Shigatoxin effector polypeptide component(s) each comprise a wild-type Shigatoxin A1 fragment.

In certain embodiments of Embodiment Sets #2 to #11, the binding regioncomprises the peptide or polypeptide shown in any one of SEQ ID NOs:83-339. In certain further embodiments, the binding region comprises orconsists essentially of the polypeptide represented by any of thefollowing: amino acids 1-245 of any one of SEQ ID NOs: 33, 64, and 65;269-513 of SEQ ID NO:40 or SEQ ID NO:80; amino acids 269-520 or 269-521of any one of SEQ ID NOs: 36, 66, and 67; amino acids 1-232, 1-233,1-234, 1-235, 1-236, 1-242, 1-243, 1-244, 1-245, 1-246, 1-252, 1-253,1-254, 1-255, or 1-256 of any one of SEQ ID NOs: 47-119 and 176-248;amino acids 269-498 or 269-499 of any one of SEQ ID NOs: 37-39, 68-79,and 81; amino acids 269-499, 269-512, 269-513, or 280-510 of any one ofSEQ ID NOs: 34, 35, 41-56, and 82.

In certain embodiments of Embodiment Sets #2 to #11, the binding regionsterically covers the carboxy-terminus of the A1 fragment region.

In certain embodiments of Embodiment Sets #2 to #11, the molecularmoiety sterically covers the carboxy-terminus of the A1 fragment region.In certain further embodiments, the molecular moiety comprises thebinding region.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention comprises a binding region and/ormolecular moiety located carboxy-terminal to the carboxy-terminus of theShiga toxin A1 fragment region. In certain further embodiments, the massof the binding region and/or molecular moiety is at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule comprises a binding region with a mass of at least 4.5 kDa, 6,kDa, 9 kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50kDa, 100 kDa, or greater, as long as the cell-targeting molecule retainsthe appropriate level of the Shiga toxin biological activity notedherein (e.g., cytotoxicity and/or intracellular routing).

In certain embodiments of Embodiment Sets #2 to #11, the binding regionis comprised within a relatively large, molecular moiety comprising suchas, e.g., a molecular moiety with a mass of at least 4.5 kDa, 6, kDa, 9kDa, 12 kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, 50 kDa, 100kDa, or greater, as long as the cell-targeting molecule retains theappropriate level of the Shiga toxin biological activity noted herein.

For certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention exhibits low cytotoxic potency (i.e.is not capable when introduced to certain positive target cell types ofexhibiting a cytotoxicity greater than 1% cell death of a cellpopulation at a cell-targeting molecule concentration of 1000 nM, 500nM, 100 nM, 75 nM, or 50 nM) and is capable when introduced to cells ofexhibiting a greater subcellular routing efficiency from anextracellular space to a subcellular compartment of an endoplasmicreticulum and/or cytosol as compared to the cytotoxicity of a referencemolecule, such as, e.g., a twenty-sixth cell-targeting molecule havingan amino-terminus and comprising the binding region and the Shiga toxineffector polypeptide which is not positioned at or proximal to theamino-terminus of the third cell-targeting molecule. In certain furtherembodiments, the twenty-sixth cell-targeting molecule does not compriseany carboxy-terminal, endoplasmic reticulum retention/retrieval signalmotif of the KDEL family.

In certain embodiments of Embodiment Sets #2 to #11, In certain furtherembodiments, the molecular moiety comprises a peptide and/or polypeptidederived from the Shiga toxin A2 fragment of a naturally occurring Shigatoxin.

The embodiments of the present invention are not intended to cover anynaturally-occurring Shiga holotoxin or Shiga toxin A Subunit. In certainembodiments of Embodiment Sets #2-11, the cell-targeting molecule of thepresent invention does not comprise a naturally occurring Shiga toxin BSubunit. In certain further embodiments, the cell-targeting molecule ofthe invention does not comprise any polypeptide comprising or consistingessentially of a functional binding domain of a native Shiga toxin Bsubunit. Rather, in certain embodiments of the cell-targeting moleculesof the invention, the Shiga toxin A Subunit derived regions arefunctionally associated with heterologous binding regions to effectuatecell-targeting.

In certain embodiments of Embodiment Sets #2 to #11, the binding regiondoes not comprise a fragment of human CD4 corresponding to amino acidresidues 19-183. In certain further embodiments, the binding region doesnot comprise a fragment of human CD4, a type-I transmembraneglycoprotein. In certain further embodiments, the binding region doesnot comprise a fragment of a human, immune cell surface co-receptor.

In certain embodiments of Embodiment Sets #2 to #11, the cell-targetingmolecule of the present invention does not comprise a carboxy-terminal,binding region comprising a fragment of an immune cell surface receptor.

In certain embodiments of Embodiment Sets #1 to #11, the Shiga toxineffector polypeptide comprises at least two, embedded or inserted,heterologous epitopes.

In certain embodiments of Embodiment Sets #1 to #11, the Shiga toxineffector polypeptide does not comprise the set of amino acid residuesubstitutions relative to a wild-type Shiga toxin A Subunit selectedfrom the following sets: (1) R248H and R251H; (2) R248G and R251G; (3)A246G, S247A, A253G, and S254A; and (4) A246G, S247A, R248G, R251G,A253G, and S254A.

In certain embodiments of Embodiment Sets #1 to #11, the Shiga toxineffector polypeptide does not comprise a deletion of the region nativelypositioned at 247-252 in a wild-type Shiga toxin A Subunit. In certainembodiments of Embodiment Sets #2-11, the Shiga toxin effectorpolypeptide does not comprise deletions of the regions nativelypositioned at 245-247 and 253-255 in a wild-type Shiga toxin A Subunit.

In certain embodiments of Embodiment Sets #1 to #11, the Shiga toxineffector polypeptide comprises one or more mutations relative to anaturally occurring A Subunit of a member of the Shiga toxin familywhich changes an enzymatic activity of the Shiga toxin effectorpolypeptide, the mutation selected from at least one amino acid residuedeletion, insertion, or substitution. In certain further embodiments,the mutation relative to the naturally occurring A Subunit reduces ofeliminates a cytotoxic activity of the Shiga toxin effector polypeptidebut the Shiga toxin effector polypeptide retains at least one otherShiga toxin effector function, such as, e.g., promoting cellularinternalization and/or directing intracellular routing to a certainsubcellular compartment(s). In certain further embodiments, the mutationrelative to the naturally occurring A Subunit is selected from at leastone amino acid residue substitution, such as, e.g., A231E, R75A, Y77S,Y114S, E167D, R170A, R176K, and/or W203A in SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3.

For certain embodiments of Embodiment Sets #1 to #11, the Shiga toxineffector polypeptide is capable of: (i) routing to a subcellularcompartment of a cell in which the Shiga toxin effector polypeptide ispresent selected from the following: cytosol, endoplasmic reticulum, andlysosome; (ii) intracellular delivery of the epitope from an earlyendosomal compartment to a proteasome of a cell in which the Shiga toxineffector polypeptide is present; and/or (iii) intracellular delivery ofthe epitope to a MHC class I molecule from an early endosomalcompartment of a cell in which the Shiga toxin effector polypeptide ispresent. In certain further embodiments, the Shiga toxin effectorpolypeptide is capable of intracellular delivery of the CD 8+ T-cellepitope for presentation by a MHC class I molecule on the surface of acell in which the Shiga toxin effector polypeptide is present.

In certain embodiments, the molecule of the present invention does notcomprise, at a position carboxy-terminal of the Shiga toxin effectorpolypeptide and/or the carboxy-terminus of the Shiga toxin A1 fragmentregion, any additional exogenous material representing an antigen and/orheterologous, CD8+, T-cell epitope-peptide.

In certain embodiments of Embodiment Sets #2 to #11, the binding regiondoes not comprise a ligand. In certain embodiments of Embodiment Sets #2to #11, the binding region does not comprise a chemokine or aTNF-related apoptosis-inducing ligand (TRAIL) nor a receptor bindingfragment thereof. In certain embodiments of Embodiment Sets #2 to #11,the binding region does not comprise a human chemokine or human TRAILnor a receptor binding fragment thereof. In embodiments of EmbodimentSets #2 to #11, the immunoglobulin-type binding region does not comprisea ligand nor a receptor binding fragment thereof. In certain embodimentsof Embodiment Sets #2 to #11, the immunoglobulin-type binding regiondoes not comprise a chemokine or a TNF-related apoptosis-inducing ligand(TRAIL) nor a receptor binding fragment thereof. In certain embodimentsof Embodiment Sets #2 to #11, the binding region does not comprise ahuman CC chemokine nor a receptor binding fragment thereof. In certainembodiments of Embodiment Sets #2 to #11, the binding region does notcomprise the human CC chemokine CCL2 (see Bose S, Cho J et al., ArchPharm Res 36: 1039-50 (2013)). In certain embodiments of Embodiment Sets#2 to #11, the binding region does not comprise the human, CC chemokineCCL2, nor a receptor binding fragment thereof, and a carboxy-terminal,Shiga toxin effector polypeptide consisting of amino acids 75-247 ofStxA. In certain embodiments of the cell-targeting molecule of thepresent invention, the binding region does not comprise the human, CCchemokine CCL2, nor a receptor binding fragment thereof, fused to acarboxy-terminal, Shiga toxin effector polypeptide consisting of aminoacids 75-247 of StxA (SEQ ID NO:2). In embodiments of Embodiment Sets #2to #11, the binding region does not comprise the human TRAIL nor areceptor binding fragment thereof.

Among certain embodiments of the present invention is a pharmaceuticalcomposition comprising any one of the above Shiga toxin effectorpolypeptides of the present invention and/or any one of the abovecell-targeting molecules of the present invention; and at least onepharmaceutically acceptable excipient or carrier.

Among certain embodiments of the present invention is a diagnosticcomposition comprising any one of the above cell-targeting molecules ofthe present invention and a detection promoting agent. Certain furtherembodiments are cell-targeting molecules of the present inventionwherein the detection promoting agent is a heterologous epitope and thecell-targeting molecule comprises the heterologous epitope.

Beyond the Shiga toxin effector polypeptides of the present invention,cell-targeting molecules of the present invention, and compositionsthereof, polynucleotides capable of encoding a Shiga toxin effectorpolypeptide or cell-targeting molecule of the present invention arewithin the scope of the present invention, as well as expression vectorswhich comprise a polynucleotide of the present invention and host cellscomprising any polynucleotide and/or expression vector of the presentinvention. Host cells comprising an expression vector may be used, e.g.,in methods for producing a molecule of the present invention or apolypeptide component or fragment thereof by recombinant expression.

Among certain embodiments of the present invention is a method ofkilling a cell comprising the step of contacting the cell with any ofthe above cell-targeting molecules of the present invention or the abovepharmaceutical compositions of the present invention. In certainembodiments, the step of contacting the cell(s) occurs in vitro. Incertain other embodiments, the step of contacting the cell(s) occurs orin vivo. In further embodiments of the cell-killing methods, the methodis capable of selectively killing cell(s) and/or cell typespreferentially over other cell(s) and/or cell types when contacting amixture of cells which differ with respect to the extracellular presenceand/or expression level of an extracellular target biomolecule of thebinding region of the cell-targeting molecule.

The present invention further provides methods of treating diseases,disorders, and/or conditions in patients comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of a cell-targeting molecule and/or pharmaceutical composition ofthe present invention. In certain embodiments, the disease, disorder, orcondition to be treated using a method of the invention is selectedfrom: a cancer, tumor, growth abnormality, immune disorder, or microbialinfection. In certain embodiments of these methods, the cancer to betreated is selected from the group consisting of: bone cancer, breastcancer, central/peripheral nervous system cancer, gastrointestinalcancer, germ cell cancer, glandular cancer, head-neck cancer,hematological cancer, kidney-urinary tract cancer, liver cancer,lung/pleura cancer, prostate cancer, sarcoma, skin cancer, and uterinecancer. In certain embodiments of these methods, the immune disorder tobe treated is an immune disorder associated with a disease selected fromthe group consisting of: amyloidosis, ankylosing spondylitis, asthma,Crohn's disease, diabetes, graft rejection, graft-versus-host disease,Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-relateddiseases, lupus erythematosus, multiple sclerosis, polyarteritis nodosa,polyarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis,scleroderma, septic shock, Sjörgren's syndrome, ulcerative colitis, andvasculitis.

The use of any composition of matter of the present invention for thetreatment or prevention of a cancer, tumor, growth abnormality, and/orimmune disorder is within the scope of the present invention. Amongcertain embodiments of the present invention is a cell-targetingmolecule of the present invention and/or a pharmaceutical compositionthereof for the treatment or prevention of a cancer, tumor, growthabnormality, immune disorder, and/or microbial infection. Among certainembodiments of the present invention is the use of a cell-targetingmolecule of the present invention and/or pharmaceutical compositionthereof in the manufacture of a medicament for the treatment orprevention of a cancer, tumor, growth abnormality, immune disorder, ormicrobial infection.

Certain embodiments of the cell-targeting molecules of the presentinvention may be utilized for the delivery of additional exogenousmaterial into a cell physically coupled with an extracellular targetbiomolecule of the cell-targeting molecule of the invention.Additionally, the present invention provides a method for deliveringexogenous material to the inside of a cell(s) comprising contacting thecell(s), either in vitro or in vivo, with a cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention. The present invention further provides a method fordelivering exogenous material to the inside of a cell(s) in a patient,the method comprising the step of administering to the patient acell-targeting molecule of the present invention (with or withoutcytotoxic activity), wherein the target cell(s) is physically coupledwith an extracellular target biomolecule of the cell-targeting molecule.

Among certain embodiments of the present invention is a method ofdelivering into a cell a T-cell epitope capable of being presented by aMHC class I molecule of the cell, the method comprising the step ofcontacting the cell with the cell-targeting molecule of the presentinvention which is associated with a heterologous, T-cell epitope and/ora composition thereof (e.g., a pharmaceutical or diagnostic compositionof the present invention).

Among certain embodiments of the present invention is a method for“seeding” a tissue locus within a chordate, the method comprising thestep of: administering to the chordate a cell-targeting molecule of thepresent invention, a pharmaceutical composition of the presentinvention, and/or a diagnostic composition of the present invention. Incertain further embodiments, the methods of the invention for “seeding”a tissue locus are for “seeding” a tissue locus which comprises amalignant, diseased, or inflamed tissue. In certain further embodiments,the methods of the invention for “seeding” a tissue locus are for“seeding” a tissue locus which comprises the tissue selected from thegroup consisting of: diseased tissue, tumor mass, cancerous growth,tumor, infected tissue, or abnormal cellular mass. In certain furtherembodiments, the methods of the invention for “seeding” a tissue locuscomprises administering to the chordate the cell-targeting molecule ofthe invention, the pharmaceutical composition of the invention, or thediagnostic composition of the invention comprising the heterologous,T-cell epitope selected from the group consisting of: peptides notnatively presented by the target cells of the cell-targeting molecule inMHC class I complexes, peptides not natively present within any proteinexpressed by the target cell, peptides not natively present within theproteome of the target cell, peptides not natively present in theextracellular microenvironment of the site to be seeded, and peptidesnot natively present in the tumor mass or infected tissue site to betargeted.

The use of any composition of matter of the present invention for thediagnosis, prognosis, and/or characterization of a disease, disorder,and/or condition is within the scope of the present invention. Amongcertain embodiments of the present invention is a method of using acell-targeting molecule of the present invention comprising a detectionpromoting agent and/or composition of the invention (e.g. a diagnosticcomposition) for the collection of information useful in the diagnosis,prognosis, or characterization of a disease, disorder, or condition.Among certain embodiments of the present invention is the method ofdetecting a cell (or subcellular compartment thereof) using acell-targeting molecule and/or diagnostic composition of the presentinvention, the method comprising the steps of contacting a cell with thecell-targeting molecule and/or diagnostic composition and detecting thepresence of said cell-targeting molecule and/or diagnostic composition.In certain embodiments, the step of contacting the cell(s) occurs invitro. In certain embodiments, the step of contacting the cell(s) occursin vivo. In certain embodiments, the step of detecting the cell(s)occurs in vitro. In certain embodiments, the step of detecting thecell(s) occurs in vivo. In certain further embodiments, the methodinvolves the detection of the location of the cell-targeting molecule inan organism using one or more imaging procedures after theadministration of the cell-targeting molecule to said organism. Forexample, cell-targeting molecules of the invention which incorporatedetection promoting agents as described herein may be used tocharacterize diseases as potentially treatable by a relatedpharmaceutical composition of the present invention. For example,certain cell-targeting molecules of the present invention andcompositions thereof (e.g. pharmaceutical compositions and diagnosticcompositions of the present invention), and methods of the presentinvention may be used to determine if a patient belongs to a group thatresponds to a pharmaceutical composition of the present invention. Forexample, certain cell-targeting molecules of the present invention andcompositions thereof may be used to identify cells which present adelivered heterologous epitope-peptide on a cellular surface and/or toidentify subjects containing cells which present a heterologousepitope-peptide delivered by a cell-targeting molecule of the presentinvention.

Among certain embodiments of the present invention is a method ofproducing a molecule of the present invention, the method comprising thestep of purifying the molecule of the invention or a polypeptidecomponent of thereof using a bacterial cell-wall protein domaininteraction, such as, e.g., protein L from P. magnus or derivatives andbinding domain fragments thereof. In certain further embodiments, thepurifying step of the method involves the Shiga toxin effectorpolypeptide comprising or consisting essentially of any one of thepolypeptides shown in SEQ ID NOs: 6-32 and 340-383. In certain furtherembodiments, the purifying step of the method involves thecell-targeting molecule comprising or consisting essentially of any oneof the polypeptides shown in SEQ ID NOs: 43-82 and 439-513.

Certain embodiments of the Shiga toxin effector polypeptides of thepresent invention may be utilized as an immunogen or as a component ofan immunogen for the immunization and/or vaccination of a chordate.Among certain embodiments of the present invention is a method ofimmunizing a chordate using a Shiga toxin effector polypeptide of thepresent invention, the method comprising administering a chordate theShiga toxin effector polypeptide of the invention. In certain furtherembodiments, the Shiga toxin effector polypeptide comprises or consistsessentially of any one of the polypeptides shown in SEQ ID NOs: 6-32 and340-383.

Among certain embodiments of the present invention are kits comprising acomposition of matter of the invention, and optionally, instructions foruse, additional reagent(s), and/or pharmaceutical delivery device(s).The kit may further comprise reagents and other tools for detecting acell type (e.g. a tumor cell) in a sample or in a subject.

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures. Theaforementioned elements of the invention may be individually combined orremoved freely in order to make other embodiments of the invention,without any statement to object to such synthesis or removalhereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts exemplary, Shiga toxin A Subunit effector polypeptides ofthe present invention (numbered 1-5) and cell-targeting moleculescomprising the same (e.g. “2/3” denotes either Shiga toxin effectorpolypeptide 2 or 3). The depictions of exemplary molecules in FIG. 1 arefor illustrative purposes of certain, general arrangements of thestructural features of a limited set of embodiments of the presentinvention. It is to be understood that these exemplary molecules do notintend, nor should any be construed, to be wholly definitive as to thearrangement of any structural features and/or components of a moleculeof the present invention. The relative size, location, or number offeatures shown in the schematics of FIG. 1 have been simplified. Forexample, the relative positions of embedded, heterologous epitopes anddisruptions of an endogenous, epitope regions are not fixed. Similarly,the total numbers of embedded, heterologous epitopes and disruptions ofan endogenous, epitope regions are not fixed. Certain embodiments of themolecules of the present invention comprise a plurality of disrupted,endogenous, epitope regions in a single, Shiga toxin effectorpolypeptide, such as, e.g., disruptions of four, five, six, seven,eight, nine, or more regions; wherein these disrupted, endogenous,epitope regions may be distributed throughout the Shiga toxin effectorpolypeptide, including disruptions which overlap with or are within thefurin-cleavage motif of the carboxy-terminus region of a Shiga toxin A1fragment derived region (see Table 8, infra). Certain embodiments of thepresent invention comprise disruptions of endogenous, epitope regionswhich are carboxy-terminal to the carboxy-terminus of the Shiga toxin A1fragment, or a derivative thereof, such as, e.g. at a positioncarboxy-terminal to any disrupted furin-cleavage site motif. Theschematics in FIG. 1 are not intended to accurately portray anyinformation regarding the relative sizes of molecular structures in anyembodiment of the present invention.

FIG. 2 graphically shows the protein synthesis inhibition activities ofexemplary cell-targeting molecules of the present invention in vitro andover a range of concentrations. For each sample molecule, theluminescent intensity of luciferase expressed during the assay inrelative luminescent units (RLU) was plotted over the logarithm to base10 of the concentration of the cell-targeting molecule tested inpicomolar. These exemplary cell-targeting molecules exhibited ribosomeinhibition activities comparable to a “control” cell-targeting molecule,whose Shiga toxin effector polypeptide component consisted of awild-type Shiga toxin A Subunit fragment except for it comprised adisrupted, furin-cleavage site at the carboxy-terminus of its Shigatoxin A1 fragment region (SLT-1A-FR (SEQ ID NO:5)).

FIG. 2 shows that exemplary alterations to naturally occurring, Shigatoxin A Subunit polypeptides, such as, e.g., de-immunizing substitutionsand embedded, heterologous, T-cell epitopes, did not significantlyimpair Shiga toxin catalytic activity.

FIGS. 3-7 and 9-10 graphically show that exemplary cell-targetingmolecules of the present invention SLT-1A-combo(n)::scFv-(n) exhibitedcell-targeted cytotoxicity comparable to “control” cell-targetingmolecules SLT-1A-FR::scFv-(n), whose Shiga toxin effector polypeptidecomponent consisted of a wild-type Shiga toxin A Subunit fragment exceptfor it comprised a disrupted, furin-cleavage site at thecarboxy-terminus of its Shiga toxin A1 fragment region (SLT-1A-FR (SEQID NO:5)). The percent viability of target positive cells for each celltype was plotted over the logarithm to base 10 of the cell-targetingmolecule concentration administered to the respective cells.

FIG. 3 graphically shows that the exemplary cell-targeting moleculeSLT-1A-combo7::scFv-1 (SEQ ID NO:44) exhibited cytotoxicity to two,different cell-types comparable to a “control” cell-targeting moleculeSLT-1A-FR::scFv-1 (SEQ ID NO:34).

FIG. 4 graphically shows that the exemplary cell-targeting moleculeSLT-1A-combo7::scFv-1 (SEQ ID NO:44) exhibited cytotoxicity to atarget-positive cell type comparable to a “control” cell-targetingmolecule SLT-1A-FR::scFv-1 (SEQ ID NO:34). The specificity ofcell-targeting was shown by using the same assay with a cell linenegative for cell-surface expression of a target biomolecule of scFv-1.In FIG. 4 for cell-line H, the percent viability of target negativecells was plotted over the logarithm to base 10 of the cell-targetingmolecule concentration administered to the cells. FIG. 4 shows that thecell-targeting molecule SLT-1A-combo7::scFv-1 (SEQ ID NO:44) did notexhibit cytotoxicity to a target negative cell type at theconcentrations tested.

FIG. 5 graphically shows that the exemplary cell-targeting moleculesSLT-1A-combo10::scFv-1 (SEQ ID NO:47), SLT-1A-combo16::scFv-1 (SEQ IDNO:52), and SLT-1A-combo19::scFv-1 (SEQ ID NO:55) exhibited cytotoxicityto two, different cell-types comparable to a “control” cell-targetingmolecule SLT-1A-FR::scFv-1 (SEQ ID NO:34).

FIG. 6 graphically shows that the exemplary cell-targeting moleculeSLT-1A-combo17::scFv-1 (SEQ ID NO:53) exhibited cytotoxicity tocell-line A comparable to a “control” cell-targeting moleculeSLT-1A-FR::scFv-1 (SEQ ID NO:34); whereas SLT-1A-combo17::scFv-1 (SEQ IDNO:53) exhibited attenuated cytotoxicity to cell-line B as compared tothe control. FIG. 6 shows that the cell-targeting moleculeSLT-1A-combo18::scFv-1 (SEQ ID NO:54) did not exhibit cytotoxicity toboth cell-types tested at concentrations up to 100 nM. The cytotoxicityresults from this assay for an untargeted, wild-type Shiga toxin A1fragment in lieu of a cell-targeting molecule are shown as well.

FIG. 7 graphically shows that the exemplary cell-targeting moleculeSLT-1A-combo2::scFv-2 (SEQ ID NO:58) exhibited cytotoxicity tocell-lines B and G comparable to a “control” cell-targeting moleculeSLT-1A-FR::scFv-2 (SEQ ID NO:35); whereas SLT-1A-combo2::scFv-2 (SEQ IDNO:58) exhibited slightly attenuated cytotoxicity to cell-line A ascompared to the control. FIG. 7 shows that the cell-targeting moleculeSLT-1A-combo13::scFv-2 (SEQ ID NO:62) exhibited strongly attenuatedcytotoxicity to the three cell-types tested as compared to the control.

FIG. 8 graphically shows that exemplary cell-targeting molecules of thepresent invention scFv-3::SLT-1A-combo5 (SEQ ID NO:64) andscFv-3::SLT-1A-combo6 (SEQ ID NO:65) exhibited cell-targetedcytotoxicity to two, different, target positive cell types comparable toa “control” cell-targeting molecule scFv-3::SLT-1A-WT (SEQ ID NO:33),whose Shiga toxin A Subunit component was a wild-type Shiga toxin ASubunit fragment. The percent viability of target positive cells fortwo, different, cell types was plotted over the logarithm to base 10 ofthe cell-targeting molecule concentration administered to the cells. Forcell-line B, both scFv-3::SLT-1A-combo5 (SEQ ID NO:64) andscFv-3::SLT-1A-combo6 (SEQ ID NO:65) exhibited cytotoxic potency asmeasured by CD₅₀ values lesser than an equivalent level of the control.The specificity of cell-targeting was shown by using the same assay witha cell line negative for cell-surface expression of a target biomoleculeof scFv-3. In FIG. 8 for cell-line A, the percent viability of targetnegative cells was plotted over the logarithm to base 10 of thecell-targeting molecule concentration administered to the cells. FIG. 8shows that the cell-targeting molecules scFv-3::SLT-1A-combo5 (SEQ IDNO:64) and scFv-3::SLT-1A-combo6 (SEQ ID NO:65) exhibited untargetedcytotoxicity to a target negative cell type comparable to the control.

FIG. 9 graphically shows that the exemplary cell-targeting moleculesSLT-1A-combo7::scFv-4 (SEQ ID NO:66) and SLT-1A-combo14::scFv-4 (SEQ IDNO:67) exhibited cell-targeted cytotoxicity to a target positive celltype comparable to a “control” cell-targeting molecule SLT-1A-FR::scFv-4(SEQ ID NO:36). The specificity of cell-targeting was shown by using thesame assay with a cell line negative for cell-surface expression of atarget biomolecule of scFv-4. In FIG. 9 for cell-line E, the percentviability of target negative cells was plotted over the logarithm tobase 10 of the cell-targeting molecule concentration administered to thecells. FIG. 9 shows that the cell-targeting moleculesSLT-1A-combo7::scFv-4 (SEQ ID NO:66) and SLT-1A-combo14::scFv-4 (SEQ IDNO:67) exhibited untargeted cytotoxicity to a target negative cell typecomparable to the control.

FIG. 10 graphically shows that the exemplary cell-targeting moleculeSLT-1A-combo8::scFv-5 (SEQ ID NO:69) exhibited cell-targetedcytotoxicity to a target positive cell type comparable to a “control”cell-targeting molecule SLT-1A-FR::scFv-5 (SEQ ID NO:37). Thecell-targeting molecule SLT-1A-combo9::scFv-5 (SEQ ID NO:70) exhibitedattenuated cytotoxicity to this cell line as compared to the control,and SLT-1A-combo11::scFv-5 (SEQ ID NO:71) exhibited very low cytotoxicpotency compared to the control.

FIGS. 11-12 graphically show the caspase activity induced by exemplarycell-targeting molecules of the present inventionSLT-1A-combo(n)::scFv-1 as compared to the “control” cell-targetingmolecule SLT-1A-FR::scFv-(n), whose Shiga toxin effector polypeptidecomponent consisted of a wild-type Shiga toxin A Subunit fragment exceptfor it comprised a disrupted, furin-cleavage site at thecarboxy-terminus of its Shiga toxin A1 fragment region (SLT-1A-FR (SEQID NO:5)). The percent caspase activity was plotted over the logarithmto base 10 of the cell-targeting molecule concentration administered tothe cells. FIGS. 11-12 show that the exemplary, cell-targeting moleculesSLT-1A-combo7::scFv-1 (SEQ ID NO:44), SLT-1A-combo14::scFv-1 (SEQ IDNO:50), and SLT-1A-combo7::scFv-7 (SEQ ID NO: 81) induced caspaseactivity comparable to a control cell-targeting molecule for at leastone cell line tested.

FIG. 13 shows the relative antigenicities of exemplary cell-targetingmolecules of the present invention and a control cell-targeting moleculeby Western blot analysis under denaturing conditions using three,different antibodies recognizing Shiga toxin A1 fragments. FIG. 13 showpictures of multiple replicate gels and membranes. The first lane marked“MW Marker” shows the migration pattern of a protein molecular weightladder, and the approximate size of each ladder protein band is labeledin kiloDaltons (kDa). The samples loaded and run in lanes numbered 1-4are indicated in the figure legend: #1) SLT-1A::combo7::scFv-1 (SEQ IDNO:44); #2) SLT-1A-FR::scFv-1 (SEQ ID NO:34); #3 SLT-1A::combo14::scFv-1(SEQ ID NO:50); and SLT-1A::combo10::scFv-1 (SEQ ID NO:47). The toppanel shows pictures of a coomassie-stained replicate gel; the secondpanel (from the top) shows pictures of replicate membrane probed withα-SLT-1A pAb1, third panel (from the top) shows pictures of a replicatemembrane probed with α-SLT-1A pAb2, and the last panel (from the top)shows pictures of a replicate membrane probed with α-StxA mAb1. FIG. 13shows that the exemplary cell-targeting molecules SLT-1A-combo7::scFv-1(SEQ ID NO:44), SLT-1A-combo10::scFv-1 (SEQ ID NO:47), andSLT-1A-combo14::scFv-1 (SEQ ID NO:50) each have reduced antigenicity inthis assay compared to the reference molecule SLT-1A-FR::scFv-1 (SEQ IDNO:34).

FIG. 14 graphically shows the relative antigenicities of exemplarycell-targeting molecules of the present invention and a controlcell-targeting molecule by ELISA analysis using two, differentantibodies recognizing Shiga toxin A1 fragments. The normalized ELISAabsorbance signal is graphed as a percentage of the control moleculeSLT-1A-FR::scFv-1 (SEQ ID NO:34). FIG. 14 shows that the exemplarycell-targeting molecules SLT-1A-combo7::scFv-1 (SEQ ID NO:44),SLT-1A-combo10::scFv-1 (SEQ ID NO:47), and SLT-1A-combo14::scFv-1 (SEQID NO:50) each have reduced antigenicity in this assay compared to thecontrol.

FIGS. 15-16 graphically show the relative immunogenicities of exemplarycell-targeting molecules of the present invention measured from seracollected from a mammalian model and using in-solution ELISA assays todetect in the serum of each animal the quantity of antibodies whichrecognize an administered Shiga toxin A Subunit derived molecule. Thereference cell-targeting molecules used for relative comparisons wereSLT-1A-FR::scFv-(n), whose Shiga toxin effector polypeptide componentconsisted of the furin-cleavage resistant, Shiga toxin effectorpolypeptide SLT-1A-FR (SEQ ID NO:5). For each animal treatment groupadministered a cell-targeting molecule of the present invention, thepercentage values of the SLT-1A-FR::scFv-(n) reference moleculetreatment group was calculated by dividing the average ELISA signal forall the subjects in the cell-targeting molecule treatment group at agiven time-point with the average ELISA signal of the subjects in theSLT-1A-FR::scFv-(n) reference treatment group. The percentage of thereference for each experimental treatment group was graphed on theY-axis, and the day of serum collection was graphed on the X-axis. Thesymbols in FIGS. 15-16 represent the average signal for individualsubjects in the indicated group, and the error bars indicate thestandard error of the mean for the subjects in the group at theindicated time-point. FIGS. 15-16 show that the exemplary cell-targetingmolecules SLT-1A-combo1::scFv-1 (SEQ ID NO:43), SLT-1A-combo7::scFv-1(SEQ ID NO:44), SLT-1A-combo10::scFv-1 (SEQ ID NO:47),SLT-1A-combo12::scFv-1 (SEQ ID NO:49), SLT-1A-combo15::scFv-1 (SEQ IDNO:51), SLT-1A-combo16::scFv-1 (SEQ ID NO: 52), SLT-1A-combo19::scFv-1(SEQ ID NO:55), SLT-1A-combo10::scFv-2 (SEQ ID NO:61), andSLT-1A-combo22::scFv-2 (SEQ ID NO:63) exhibited reduced immunogenicityin this assay relative to the appropriate, reference moleculeSLT-1A-FR::scFv-(n) (SEQ ID NO:34 or 35).

FIG. 17 shows the furin-cleavage resistance of the cell-targetedmolecule SLT-1A-FR::scFv-9, which comprised a Shiga toxin effectorpolypeptide comprising a disrupted furin-cleavage motif, as compared toa nearly identical, cytotoxic, cell-targeted molecule comprising awild-type Shiga toxin A1 fragment with a wild-type furin-cleavage site(SLT-1A-WT::scFv-9). FIG. 17 shows a coomassie-stained, polyacrylamidegel after electrophoresis of protein samples treated with eitherpurified, recombinant, human furin or various negative controlconditions. The lanes of the gel are numbered, and the figure legendindicates pre-treatment conditions of each cell-targeted molecule sampleprior to loading sample to the gel: the temperature in degrees Celsius(° C.), the pre-treatment duration in hours, and whether any furin wasadded by denoting the amount of furin activity units added (U) permicrogram (μg) of cell-targeted molecule (labeled “U/μg furin”) or “nofurin” for zero U/μg furin added. The first lane marked “MW Marker”shows the migration pattern of a protein molecular weight ladder, andthe approximate size of each ladder protein band is labeled inkiloDaltons (kDa). The figure legend indicates which Shiga toxineffector region was present in each cell-targeted molecule sample perlane, either 1) a wild-type furin site (WT) or 2) a disrupted furinmotif (FR). The treated samples were subjected to 0.5 furin activityunits per microgram of cell-targeted molecule (U/pg furin) at 30° C. for30 hours. FIG. 17 shows SLT-1A-FR::scFv-9 was resistant to 0.5 furinactivity units per microgram of SLT-1A-FR::scFv-9 at 30° C.

FIG. 18 graphically shows the specific binding of an exemplary,cell-targeting molecule of the present invention (SEQ ID NO:82) totarget positive cells as compared to target negative cells. The amountof cell-targeting molecule binding to cells was calculated as anintegrated, mean fluorescence intensity (iMFI) and graphed versus theconcentration of the cell-targeting molecule. Curve fitting of the datawas used to produce the lines for the two, target-positive cell-typestested.

FIG. 19 graphically shows the change in human tumor burdens over timefor groups of subjects in a murine xenograft model after receivingeither an exemplary, cell-targeting molecule of the present invention ora vehicle-only control sample. The tumor burden measured as whole bodybioluminescence in photons/second was graphed versus time (dayspost-tumor implant). Administration of the cell-targeting moleculeSLT1-A-combo7::αCD38-scFv-1 (SEQ ID NO:82) prevented the increase intumor burden observed for the vehicle only control group.

DETAILED DESCRIPTION

The present invention is described more fully hereinafter usingillustrative, non-limiting embodiments, and references to theaccompanying figures. This invention may, however, be embodied in manydifferent forms and should not be construed as to be limited to theembodiments set forth below. Rather, these embodiments are provided sothat this disclosure is thorough and conveys the scope of the inventionto those skilled in the art.

In order that the present invention may be more readily understood,certain terms are defined below. Additional definitions may be foundwithin the detailed description of the invention.

As used in the specification and the appended claims, the terms “a,”“an” and “the” include both singular and the plural referents unless thecontext clearly dictates otherwise.

As used in the specification and the appended claims, the term “and/or”when referring to two species, A and B, means at least one of A and B.As used in the specification and the appended claims, the term “and/or”when referring to greater than two species, such as A, B, and C, meansat least one of A, B, or C, or at least one of any combination of A, B,or C (with each species in singular or multiple possibility).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

Throughout this specification, the term “including” is used to mean“including but not limited to.” “Including” and “including but notlimited to” are used interchangeably.

The term “amino acid residue” or “amino acid” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.The term “polypeptide” includes any polymer of amino acids or amino acidresidues. The term “polypeptide sequence” refers to a series of aminoacids or amino acid residues which physically comprise a polypeptide. A“protein” is a macromolecule comprising one or more polypeptides orpolypeptide “chains.” A “peptide” is a small polypeptide of sizes lessthan about a total of 15 to 20 amino acid residues. The term “amino acidsequence” refers to a series of amino acids or amino acid residues whichphysically comprise a peptide or polypeptide depending on the length.Unless otherwise indicated, polypeptide and protein sequences disclosedherein are written from left to right representing their order from anamino-terminus to a carboxy-terminus.

The terms “amino acid,” “amino acid residue,” “amino acid sequence,” orpolypeptide sequence include naturally occurring amino acids (includingL and D isosteriomers) and, unless otherwise limited, also include knownanalogs of natural amino acids that can function in a similar manner asnaturally occurring amino acids, such as selenocysteine, pyrrolysine,N-formylmethionine, gamma-carboxyglutamate, hydroxyprolinehypusine,pyroglutamic acid, and selenomethionine. The amino acids referred toherein are described by shorthand designations as follows in Table A:

TABLE A Amino Acid Nomenclature Name 3-letter 1-letter Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid or Aspartate Asp DCysteine Cys C Glutamic Acid or Glutamate Glu E Glutamine Gln Q GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The phrase “conservative substitution” with regard to an amino acidresidue of a peptide, peptide region, polypeptide region, protein, ormolecule refers to a change in the amino acid composition of thepeptide, peptide region, polypeptide region, protein, or molecule thatdoes not substantially alter the function and structure of the overallpeptide, peptide region, polypeptide region, protein, or molecule (seeCreighton, Proteins: Structures and Molecular Properties (W. H. Freemanand Company, New York (2nd ed., 1992))).

For purposes of the present invention, the phrase “derived from” whenreferring to a polypeptide or polypeptide region means that thepolypeptide or polypeptide region comprises amino acid sequencesoriginally found in a “parental” protein and which may now comprisecertain amino acid residue additions, deletions, truncations,rearrangements, or other alterations relative to the originalpolypeptide or polypeptide region as long as a certain function(s) and astructure(s) of the “parental” molecule are substantially conserved. Theskilled worker will be able to identify a parental molecule from which apolypeptide or polypeptide region was derived using techniques known inthe art, e.g., protein sequence alignment software.

For purposes of the claimed invention and with regard to a Shiga toxinpolypeptide sequence or Shiga toxin derived polypeptide, the term“wild-type” generally refers to a naturally occurring, Shiga toxinprotein sequence(s) found in a living species, such as, e.g., apathogenic bacterium, wherein that Shiga toxin protein sequence(s) isone of the most frequently occurring variants. This is in contrast toinfrequently occurring Shiga toxin protein sequences that, while stillnaturally occurring, are found in less than one percent of individualorganisms of a given species when sampling a statistically powerfulnumber of naturally occurring individual organisms of that species whichcomprise at least one Shiga toxin protein variant. A clonal expansion ofa natural isolate outside its natural environment (regardless of whetherthe isolate is an organism or molecule comprising biological sequenceinformation) does not alter the naturally occurring requirement as longas the clonal expansion does not introduce new sequence variety notpresent in naturally occurring populations of that species and/or doesnot change the relative proportions of sequence variants to each other.

The terms “associated,” “associating,” “linked,” or “linking” withregard to the claimed invention refers to the state of two or morecomponents of a molecule being joined, attached, connected, or otherwisecoupled to form a single molecule or the act of making two moleculesassociated with each other to form a single molecule by creating anassociation, linkage, attachment, and/or any other connection betweenthe two molecules. For example, the term “linked” may refer to two ormore components associated by one or more atomic interactions such thata single molecule is formed and wherein the atomic interactions may becovalent and/or non-covalent. Non-limiting examples of covalentassociations between two components include peptide bonds andcysteine-cysteine disulfide bonds. Non-limiting examples of non-covalentassociations between two molecular components include ionic bonds.

For purposes of the present invention, the term “linked” refer to two ormore molecular components associated by one or more atomic interactionssuch that a single molecule is formed and wherein the atomicinteractions includes at least one covalent bond. For purposes of thepresent invention, the term “linking” refers to the act of creating alinked molecule as described above.

For purposes of the present invention, the term “fused” refers to two ormore proteinaceous components associated by at least one covalent bondwhich is a peptide bond, regardless of whether the peptide bond involvesthe participation of a carbon atom of a carboxyl acid group or involvesanother carbon atom, such as, e.g., the α-carbon, β-carbon, γ-carbon,σ-carbon, etc. Non-limiting examples of two proteinaceous componentsfused together include, e.g., an amino acid, peptide, or polypeptidefused to a polypeptide via a peptide bond such that the resultingmolecule is a single, continuous polypeptide. For purposes of thepresent invention, the term “fusing” refers to the act of creating afused molecule as described above, such as, e.g., a fusion proteingenerated from the recombinant fusion of genetic regions which whentranslated produces a single proteinaceous molecule.

The symbol “::” means the polypeptide regions before and after it arephysically linked together to form a continuous polypeptide.

As used herein, the terms “expressed,” “expressing,” or “expresses,” andgrammatical variants thereof, refer to translation of a polynucleotideor nucleic acid into a protein. The expressed protein may remainintracellular, become a component of the cell surface membrane or besecreted into an extracellular space.

As used herein, cells which express a significant amount of anextracellular target biomolecule at least one cellular surface are“target positive cells” or “target+cells” and are cells physicallycoupled to the specified, extracellular target biomolecule.

As used herein, the symbol “a” is shorthand for an immunoglobulin-typebinding region capable of binding to the biomolecule following thesymbol. The symbol “a” is used to refer to the functional characteristicof an immunoglobulin-type binding region based on its ability to bind tothe biomolecule following the symbol with a binding affinity describedby a dissociation constant (K_(D)) of 10⁻⁵ or less.

As used herein, the term “heavy chain variable (V_(H)) domain” or “lightchain variable (V_(L)) domain” respectively refer to any antibody V_(H)or V_(L) domain (e.g. a human V_(H) or V_(L) domain) as well as anyderivative thereof retaining at least qualitative antigen bindingability of the corresponding native antibody (e.g. a humanized V_(H) orV_(L) domain derived from a native murine V_(H) or V_(L) domain). AV_(H) or V_(L) domain consists of a “framework” region interrupted bythe three CDRs or ABRs. The framework regions serve to align the CDRs orABRs for specific binding to an epitope of an antigen. Fromamino-terminus to carboxy-terminus, both V_(H) and V_(L) domainscomprise the following framework (FR) and CDR regions or ABR regions:FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4; or, similarly, FR1, ABR1, FR2,ABR2, FR3, ABR3, and FR4. As used herein, the terms “HCDR1,” “HCDR2,” or“HCDR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V_(H)domain, and the terms “LCDR1,” “LCDR2,” and “LCDR3” are used to refer toCDRs 1, 2, or 3, respectively, in a V_(L) domain. As used herein, theterms “HABR1,” “HABR2,” or “HABR3” are used to refer to ABRs 1, 2, or 3,respectively, in a V_(H) domain, and the terms “LABR1,” “LABR2,” or“LABR3” are used to refer to CDRs 1, 2, or 3, respectively, in a V_(L)domain. For camelid V_(H)H fragments, IgNARs of cartilaginous fish,V_(NAR) fragments, certain single domain antibodies, and derivativesthereof, there is a single, heavy chain variable domain comprising thesame basic arrangement: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Asused herein, the terms “HCDR1,” “HCDR2,” or “HCDR3” may be used to referto CDRs 1, 2, or 3, respectively, in a single heavy chain variabledomain.

For purposes of the present invention, the term “effector” meansproviding a biological activity, such as cytotoxicity, biologicalsignaling, enzymatic catalysis, subcellular routing, and/orintermolecular binding resulting in an allosteric effect(s) and/or therecruitment of one or more factors.

For purposes of the present invention, the phrases “Shiga toxin effectorpolypeptide,” “Shiga toxin effector polypeptide region,” and “Shigatoxin effector region” refer to a polypeptide or polypeptide regionderived from at least one Shiga toxin A Subunit of a member of the Shigatoxin family wherein the polypeptide or polypeptide region is capable ofexhibiting at least one Shiga toxin function. For example, SEQ ID NOs:8, 11-27, 29-32, 348, 356-358, 363, 370-371, 373, 378-438 are derivedfrom StxA and SLT-1A.

For purposes of the present invention, a Shiga toxin effector functionis a biological activity conferred by a polypeptide region derived froma Shiga toxin A Subunit. Non-limiting examples of Shiga toxin effectorfunctions include promoting cell entry; lipid membrane deformation;promoting cellular internalization; stimulating clathrin-mediatedendocytosis; directing intracellular routing to various intracellularcompartments such as, e.g., the Golgi, endoplasmic reticulum, andcytosol; directing intracellular routing with a cargo; inhibiting aribosome function(s); catalytic activities, such as, e.g., N-glycosidaseactivity and catalytically inhibiting ribosomes; reducing proteinsynthesis, inducing caspase activity, activating effector caspases,effectuating cytostatic effects, and cytotoxicity. Shiga toxin catalyticactivities include, for example, ribosome inactivation, proteinsynthesis inhibition, N-glycosidase activity, polynucleotide:adenosineglycosidase activity, RNAase activity, and DNAase activity. Shiga toxinsare ribosome inactivating proteins (RIPs). RIPs can depurinate nucleicacids, polynucleosides, polynucleotides, rRNA, ssDNA, dsDNA, mRNA (andpolyA), and viral nucleic acids (see e.g., Barbieri L et al., Biochem J286: 1-4 (1992); Barbieri L et al., Nature 372: 624 (1994); Ling J etal., FEBS Lett 345: 143-6 (1994); Barbieri L et al., Biochem J319:507-13 (1996); Roncuzzi L, Gasperi-Campani A, FEBS Lett 392: 16-20(1996); Stirpe F et al., FEBS Lett 382: 309-12 (1996); Barbieri L etal., Nucleic Acids Res 25: 518-22 (1997); Wang P, Tumer N, Nucleic AcidsRes 27: 1900-5 (1999); Barbieri L et al., Biochim Biophys Acta 1480:258-66 (2000); Barbieri L et al., J Biochem 128: 883-9 (2000); BrigottiM et al., Toxicon 39: 341-8 (2001); Brigotti M et al., FASEB J 16:365-72 (2002); Bagga S et al., J Biol Chem 278: 4813-20 (2003); Picard Det al., J Biol Chem 280: 20069-75 (2005)). Some RIPs show antiviralactivity and superoxide dismutase activity (Erice A et al., AntimicrobAgents Chemother 37: 835-8 (1993); Au T et al., FEBS Lett 471: 169-72(2000); Parikh B, Tumer N, Mini Rev Med Chem 4: 523-43 (2004); Sharma Net al., Plant Physiol 134: 171-81 (2004)). Shiga toxin catalyticactivities have been observed both in vitro and in vivo. Non-limitingexamples of assays for Shiga toxin effector activity measure variousactivities, such as, e.g., protein synthesis inhibitory activity,depurination activity, inhibition of cell growth, cytotoxicity,supercoiled DNA relaxation activity, and nuclease activity.

As used herein, the retention of Shiga toxin effector function refers tobeing capable of exhibiting a level of Shiga toxin functional activity,as measured by an appropriate quantitative assay with reproducibility,comparable to a wild-type, Shiga toxin effector polypeptide control(e.g. a Shiga toxin A1 fragment) or cell-targeting molecule comprising awild-type Shiga toxin effector polypeptide (e.g. a Shiga toxin A1fragment) under the same conditions. For the Shiga toxin effectorfunction of ribosome inactivation or ribosome inhibition, retained Shigatoxin effector function is exhibiting an IC₅₀ of 10,000 pM or less in anin vitro setting, such as, e.g., by using an assay known to the skilledworker and/or described herein. For the Shiga toxin effector function ofcytotoxicity in a target positive cell-kill assay, retained Shiga toxineffector function is exhibiting a CD₅₀ of 1,000 nM or less, depending onthe cell type and its expression of the appropriate extracellular targetbiomolecule, as shown, e.g., by using an assay known to the skilledworker and/or described herein.

For purposes of the claimed invention, the term “equivalent” with regardto ribosome inhibition means an empirically measured level of ribosomeinhibitory activity, as measured by an appropriate quantitative assaywith reproducibility, which is reproducibly within 10% or less of theactivity of the reference molecule (e.g., the second cell-targetingmolecule or third cell-targeting molecule) under the same conditions.

For purposes of the claimed invention, the term “equivalent” with regardto cytotoxicity means an empirically measured level of cytotoxicity, asmeasured by an appropriate quantitative assay with reproducibility,which is reproducibly within 10% or less of the activity of thereference molecule (e.g., the second cell-targeting molecule or thirdcell-targeting molecule) under the same conditions.

As used herein, the term “attenuated” with regard to cytotoxicity meansa molecule exhibits or exhibited a CD₅₀ between 10-fold to 100-fold of aCD₅₀ exhibited by a reference molecule under the same conditions.

Inaccurate IC₅₀ and CD₅₀ values should not be considered whendetermining a level of Shiga toxin effector function activity. For somesamples, accurate values for either IC₅₀ or CD₅₀ might be unobtainabledue to the inability to collect the required data points for an accuratecurve fit. For example, theoretically, neither an IC₅₀ nor CD₅₀ can bedetermined if greater than 50% ribosome inhibition or cell death,respectively, does not occur in a concentration series for a givensample. Data insufficient to accurately fit a curve as described in theanalysis of the data from exemplary Shiga toxin effector functionassays, such as, e.g., assays described in the Examples below, shouldnot be considered as representative of actual Shiga toxin effectorfunction.

A failure to detect activity in Shiga toxin effector function may be dueto improper expression, polypeptide folding, and/or protein stabilityrather than a lack of cell entry, subcellular routing, and/or enzymaticactivity. Assays for Shiga toxin effector functions may not require muchpolypeptide of the invention to measure significant amounts of Shigatoxin effector function activity. To the extent that an underlying causeof low or no effector function is determined empirically to relate toprotein expression or stability, one of skill in the art may be able tocompensate for such factors using protein chemistry and molecularengineering techniques known in the art, such that a Shiga toxinfunctional effector activity may be restored and measured. As examples,improper cell-based expression may be compensated for by using differentexpression control sequences; and improper polypeptide folding and/orstability may benefit from stabilizing terminal sequences, orcompensatory mutations in non-effector regions which stabilize the threedimensional structure of the molecule.

Certain Shiga toxin effector functions are not easily measurable, e.g.subcellular routing functions. For example, there is no routine,quantitative assay to distinguish whether the failure of a Shiga toxineffector polypeptide to be cytotoxic and/or deliver a heterologousepitope is due to improper subcellular routing, but at a time when testsare available, then Shiga toxin effector polypeptides may be analyzedfor any significant level of subcellular routing as compared to theappropriate wild-type Shiga toxin effector polypeptide. However, if aShiga toxin effector polypeptide component of a cell-targeting moleculeof the present invention exhibits cytotoxicity comparable or equivalentto a wild-type Shiga toxin A Subunit construct, then the subcellularrouting activity level is inferred to be comparable or equivalent,respectively, to the subcellular routing activity level of a wild-typeShiga toxin A Subunit construct at least under the conditions tested.

When new assays for individual Shiga toxin functions become available,Shiga toxin effector polypeptides and/or cell-targeting moleculescomprising Shiga toxin effector polypeptides may be analyzed for anylevel of those Shiga toxin effector functions, such as a being within1000-fold or 100-fold or less the activity of a wild-type Shiga toxineffector polypeptide or exhibiting 3-fold to 30-fold or greater activityas compared to a functional knockout, Shiga toxin effector polypeptide.

Sufficient subcellular routing may be merely deduced by observing amolecule's cytotoxic activity levels in cytotoxicity assays, such as,e.g., cytotoxicity assays based on T-cell epitope presentation or basedon a toxin effector function involving a cytosolic and/or endoplasmicreticulum-localized, target substrate.

As used herein, the retention of “significant” Shiga toxin effectorfunction refers to a level of Shiga toxin functional activity, asmeasured by an appropriate quantitative assay with reproducibilitycomparable to a wild-type Shiga toxin effector polypeptide control (e.g.a Shiga toxin A1 fragment). For in vitro ribosome inhibition,significant Shiga toxin effector function is exhibiting an IC₅₀ of 300pM or less depending on the source of the ribosomes used in the assay(e.g. a bacterial, archaeal, or eukaryotic (algal, fungal, plant, oranimal) source). This is significantly greater inhibition as compared tothe approximate IC₅₀ of 100,000 pM for the catalytically disruptedSLT-1A 1-251 double mutant (Y77S/E167D). For cytotoxicity in atarget-positive cell-kill assay in laboratory cell culture, significantShiga toxin effector function is exhibiting a CD₅₀ of 100, 50, 30 nM, orless, depending on the target biomolecule(s) of the binding region andthe cell type, particularly that cell type's expression and/orcell-surface representation of the appropriate extracellular targetbiomolecule(s) and/or the extracellular epitope(s) targeted by themolecule being evaluated. This is significantly greater cytotoxicity tothe appropriate, target-positive cell population as compared to a Shigatoxin A Subunit alone (or a wild-type Shiga toxin A1 fragment), withouta cell targeting binding region, which has a CD₅₀ of 100-10,000 nM,depending on the cell line.

For purposes of the present invention and with regard to the Shiga toxineffector function of a molecule of the present invention, the term“reasonable activity” refers to exhibiting at least a moderate level(e.g. within 11-fold to 1,000-fold) of Shiga toxin effector activity asdefined herein in relation to a molecule comprising a naturallyoccurring Shiga toxin, wherein the Shiga toxin effector activity isselected from the group consisting of: internalization efficiency,subcellular routing efficiency to the cytosol, delivered epitopepresentation by a target cell(s), ribosome inhibition, and cytotoxicity.For cytotoxicity, a reasonable level of Shiga toxin effector activityincludes being within 1,000-fold of a wild-type, Shiga toxin construct,such as, e.g., exhibiting a CD₅₀ of 500 nM or less when a wild-typeShiga toxin construct exhibits a CD₅₀ of 0.5 nM (e.g. a cell-targetingmolecule comprising a wild-type Shiga toxin A1 fragment).

For purposes of the present invention and with regard to thecytotoxicity of a molecule of the present invention, the term “optimal”refers to a level of Shiga toxin catalytic domain mediated cytotoxicitythat is within 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold of the cytotoxicity ofa molecule comprising wild-type Shiga toxin A1 fragment (e.g. a Shigatoxin A Subunit or certain truncated variants thereof) and/or anaturally occurring Shiga toxin.

It should be noted that even if the cytotoxicity of a Shiga toxineffector polypeptide is reduced relative to a wild-type Shiga toxin ASubunit or fragment thereof, in practice, applications using attenuated,Shiga toxin effector polypeptides may be equally or more effective thanusing wild-type Shiga toxin effector polypeptides because the highestpotency variants might exhibit undesirable effects which are minimizedor reduced in reduced cytotoxic-potency variants. Wild-type Shiga toxinsare very potent, being able to kill an intoxicated cell after only onetoxin molecule has reached the cytosol of the intoxicated cell orperhaps after only forty toxin molecules have been internalized into theintoxicated cell. Shiga toxin effector polypeptides with evenconsiderably reduced Shiga toxin effector functions, such as, e.g.,subcellular routing or cytotoxicity, as compared to wild-type Shigatoxin effector polypeptides may still be potent enough for practicalapplications, such as, e.g., applications involving targetedcell-killing, heterologous epitope delivery, and/or detection ofspecific cells and their subcellular compartments. In addition, certainreduced-activity Shiga toxin effector polypeptides may be particularlyuseful for delivering cargos (e.g. an additional exogenous material orT-cell epitope) to certain intracellular locations or subcellularcompartments of target cells.

The term “selective cytotoxicity” with regard to the cytotoxic activityof a molecule refers to the relative level of cytotoxicity between abiomolecule target positive cell population (e.g. a targeted cell-type)and a non-targeted bystander cell population (e.g. a biomolecule targetnegative cell-type), which can be expressed as a ratio of thehalf-maximal cytotoxic concentration (CD₅₀) for a targeted cell typeover the CD₅₀ for an untargeted cell type to provide a metric ofcytotoxic selectivity or indication of the preferentiality of killing ofa targeted cell versus an untargeted cell.

The cell surface representation and/or density of a given extracellulartarget biomolecule (or extracellular epitope of a given targetbiomolecule) may influence the applications for which certaincell-targeting molecules of the present invention may be most suitablyused. Differences in cell surface representation and/or density of agiven target biomolecule between cells may alter, both quantitativelyand qualitatively, the efficiency of cellular internalization and/orcytotoxicity potency of a given cell-targeting molecule of the presentinvention. The cell surface representation and/or density of a giventarget biomolecule can vary greatly among target biomolecule positivecells or even on the same cell at different points in the cell cycle orcell differentiation. The total cell surface representation of a giventarget biomolecule and/or of certain extracellular epitopes of a giventarget biomolecule on a particular cell or population of cells may bedetermined using methods known to the skilled worker, such as methodsinvolving fluorescence-activated cell sorting (FACS) flow cytometry.

As used herein, the terms “disrupted,” “disruption,” or “disrupting,”and grammatical variants thereof, with regard to a polypeptide region orfeature within a polypeptide refers to an alteration of at least oneamino acid within the region or composing the disrupted feature. Aminoacid alterations include various mutations, such as, e.g., a deletion,inversion, insertion, or substitution which alter the amino acidsequence of the polypeptide. Amino acid alterations also includechemical changes, such as, e.g., the alteration one or more atoms in anamino acid functional group or the addition of one or more atoms to anamino acid functional group.

As used herein, “de-immunized” means reduced antigenic and/orimmunogenic potential after administration to a chordate as compared toa reference molecule, such as, e.g., a wild-type peptide region,polypeptide region, or polypeptide. This includes a reduction in overallantigenic and/or immunogenic potential despite the introduction of oneor more, de novo, antigenic and/or immunogenic epitopes as compared to areference molecule. For certain embodiments, “de-immunized” means amolecule exhibited reduced antigenicity and/or immunogenicity afteradministration to a mammal as compared to a “parental” molecule fromwhich it was derived, such as, e.g., a wild-type Shiga toxin A1fragment. In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention is capable of exhibiting a relativeantigenicity compared to a reference molecule which is reduced by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than the antigenicityof the reference molecule under the same conditions measured by the sameassay, such as, e.g., an assay known to the skilled worker and/ordescribed herein like a quantitative ELISA or Western blot analysis. Incertain embodiments, the de-immunized, Shiga toxin effector polypeptideof the present invention is capable of exhibiting a relativeimmunogenicity compared to a reference molecule which is reduced by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater thanthe immunogenicity of the reference molecule under the same conditionsmeasured by the same assay, such as, e.g., an assay known to the skilledworker and/or described herein like a quantitative measurement ofanti-molecule antibodies produced in a mammal(s) after receivingparenteral administration of the molecule at a given time-point.

The relative immunogenicities of exemplary cell-targeting molecules weredetermined using an assay for in vivo antibody responses to thecell-targeting molecules after repeat, parenteral administrations overperiods of many.

For purposes of the present invention, the phrase “B-cell and/or CD4+T-cell de-immunized” means that the molecule has a reduced antigenicand/or immunogenic potential after administration to a mammal regardingeither B-cell antigenicity or immunogenicity and/or CD4+ T-cellantigenicity or immunogenicity.

For certain embodiments, “B-cell de-immunized” means a moleculeexhibited reduced B-cell antigenicity and/or immunogenicity afteradministration to a mammal as compared to a “parental” molecule fromwhich it was derived, such as, e.g., a wild-type Shiga toxin A1fragment. For certain embodiments, “CD4+ T-cell de-immunized” means amolecule exhibited reduced CD4 T-cell antigenicity and/or immunogenicityafter administration to a mammal as compared to a “parental” moleculefrom which it was derived, such as, e.g., a wild-type Shiga toxin A1fragment.

The term “endogenous” with regard to a B-cell epitope, CD4+ T-cellepitope, B-cell epitope region, or CD4+ T-cell epitope region in a Shigatoxin effector polypeptide refers to an epitope present in a wild-typeShiga toxin A Subunit.

For purposes of the present invention, the phrase “CD8+ T-cellhyper-immunized” means that the molecule, when present inside anucleated, chordate cell within a living chordate, has an increasedantigenic and/or immunogenic potential regarding CD8+ T-cellantigenicity or immunogenicity. Commonly, CD8+ T-cell immunizedmolecules are capable of cellular internalization to an early endosomalcompartment of a nucleated, chordate cell due either to an inherentfeature(s) or as a component of a cell-targeting molecule.

For purposes of the present invention, the term “heterologous” means ofa different source than an A Subunit of a naturally occurring Shigatoxin, e.g. a heterologous polypeptide is not naturally found as part ofany A Subunit of a native Shiga toxin. The term “heterologous” withregard to T-cell epitope or T-cell epitope-peptide component of apolypeptide of the present invention refers to an epitope or peptidesequence which did not initially occur in the polypeptide to bemodified, but which has been added to the polypeptide, whether added viathe processes of embedding, fusion, insertion, and/or amino acidsubstitution as described herein, or by any other engineering means. Theresult is a modified polypeptide comprising a T-cell epitope foreign tothe original, unmodified polypeptide, i.e. the T-cell epitope was notpresent in the original polypeptide.

The term “embedded” and grammatical variants thereof with regard to aT-cell epitope or T-cell epitope-peptide component of a polypeptide ofthe present invention refers to the internal replacement of one or moreamino acids within a polypeptide region with different amino acids inorder to generate a new polypeptide sequence sharing the same totalnumber of amino acid residues with the starting polypeptide region.Thus, the term “embedded” does not include any external, terminal fusionof any additional amino acid, peptide, or polypeptide component to thestarting polypeptide nor any additional internal insertion of anyadditional amino acid residues, but rather includes only substitutionsfor existing amino acids. The internal replacement may be accomplishedmerely by amino acid residue substitution or by a series ofsubstitutions, deletions, insertions, and/or inversions. If an insertionof one or more amino acids is used, then the equivalent number ofproximal amino acids must be deleted next to the insertion to result inan embedded T-cell epitope. This is in contrast to use of the term“inserted” with regard to a T-cell epitope contained within apolypeptide of the present invention to refer to the insertion of one ormore amino acids internally within a polypeptide resulting in a newpolypeptide having an increased number of amino acids residues comparedto the starting polypeptide.

The term “inserted” and grammatical variants thereof with regard to aT-cell epitope contained within a polypeptide of the present inventionrefers to the insertion of one or more amino acids within a polypeptideresulting in a new polypeptide sequence having an increased number ofamino acids residues compared to the starting polypeptide. The “pure”insertion of a T-cell epitope-peptide is when the resulting polypeptideincreased in length by the number of amino acid residues equivalent tothe number of amino acid residues in the entire, inserted T-cellepitope-peptide. The phrases “partially inserted,” “embedded andinserted,” and grammatical variants thereof with regard to a T-cellepitope contained within a polypeptide of the present invention, refersto when the resulting polypeptide increased in length, but by less thanthe number of amino acid residues equivalent to the length of theentire, inserted T-cell epitope-peptide. Insertions, whether “pure” or“partial,” include any of the previously described insertions even ifother regions of the polypeptide not proximal to the insertion sitewithin the polypeptide are deleted thereby resulting in a decrease inthe total length of the final polypeptide because the final polypeptidestill comprises an internal insertion of one or more amino acids of aT-cell epitope-peptide within a polypeptide region.

As used herein, the term “T-cell epitope delivering” when describing afunctional activity of a molecule means that a molecule provides thebiological activity of localizing within a cell to a subcellularcompartment that is competent to result in the proteasomal cleavage of aproteinaceous part of the molecule which comprises a T-cellepitope-peptide. The “T-cell epitope delivering” function of a moleculecan be assayed by observing the MHC presentation of a T-cellepitope-peptide cargo of the molecule on a cell surface of a cellexogenously administered the molecule or in which the assay was begunwith the cell containing the molecule in one or more of its endosomalcompartments. Generally, the ability of a molecule to deliver a T-cellepitope to a proteasome can be determined where the initial location ofthe “T-cell epitope delivering” molecule is an early endosomalcompartment of a cell, and then, the molecule is empirically shown todeliver the epitope-peptide to the proteasome of the cell. However, a“T-cell epitope delivering” ability may also be determined where themolecule starts at an extracellular location and is empirically shown,either directly or indirectly, to deliver the epitope into a cell and toproteasomes of the cell. For example, certain “T-cell epitopedelivering” molecules pass through an endosomal compartment of the cell,such as, e.g. after endocytotic entry into that cell. Alternatively,“T-cell epitope delivering” activity may be observed for a moleculestarting at an extracellular location whereby the molecule does notenter any endosomal compartment of a cell-instead the “T-cell epitopedelivering” molecule enters a cell and delivers a T-cell epitope-peptideto proteasomes of the cell, presumably because the “T-cell epitopedelivering” molecule directed its own routing to a subcellularcompartment competent to result in proteasomal cleavage of its T-cellepitope-peptide component.

For purposes of the present invention, the phrase “proximal to anamino-terminus” with reference to the position of a Shiga toxin effectorpolypeptide region of a cell-targeting molecule of the present inventionrefers to a distance wherein at least one amino acid residue of theShiga toxin effector polypeptide region is within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12 or more, e.g., up to 18-20 amino acid residues, of anamino-terminus of the cell-targeting molecule as long as thecell-targeting molecule is capable of exhibiting the appropriate levelof Shiga toxin effector functional activity noted herein (e.g., acertain level of cytotoxic potency). Thus for certain embodiments of thepresent invention, any amino acid residue(s) fused amino-terminal to theShiga toxin effector polypeptide should not reduce any Shiga toxineffector function (e.g., by sterically hindering a structure(s) near theamino-terminus of the Shiga toxin effector polypeptide region) such thata functional activity of the Shiga toxin effector polypeptide is reducedbelow the appropriate activity level required herein.

For purposes of the present invention, the phrase “more proximal to anamino-terminus” with reference to the position of a Shiga toxin effectorpolypeptide region within a cell-targeting molecule of the presentinvention as compared to another component (e.g., a cell-targeting,binding region, molecular moiety, and/or additional exogenous material)refers to a position wherein at least one amino acid residue of theamino-terminus of the Shiga toxin effector polypeptide is closer to theamino-terminus of a linear, polypeptide component of the cell-targetingmolecule of the present invention as compared to the other referencedcomponent.

For purposes of the present invention, the phrase “active enzymaticdomain derived from one A Subunit of a member of the Shiga toxin family”refers to having the ability to inhibit protein synthesis via acatalytic ribosome inactivation mechanism. The enzymatic activities ofnaturally occurring Shiga toxins may be defined by the ability toinhibit protein translation using assays known to the skilled worker,such as, e.g., in vitro assays involving RNA translation in the absenceof living cells or in vivo assays involving RNA translation in a livingcell. Using assays known to the skilled worker and/or described herein,the potency of a Shiga toxin enzymatic activity may be assessed directlyby observing N-glycosidase activity toward ribosomal RNA (rRNA), suchas, e.g., a ribosome nicking assay, and/or indirectly by observinginhibition of ribosome function and/or protein synthesis.

For purposes of the present invention, the term “Shiga toxin A1 fragmentregion” refers to a polypeptide region consisting essentially of a Shigatoxin A1 fragment and/or derived from a Shiga toxin A1 fragment of aShiga toxin.

For purposes of the present invention, the terms “terminus,”“amino-terminus,” or “carboxy-terminus” with regard to a cell-targetingmolecule refers generally to the last amino acid residue of apolypeptide chain of the cell-targeting molecule (e.g., a single,continuous polypeptide chain). A cell-targeting molecule may comprisemore than one polypeptides or proteins, and, thus, a cell-targetingmolecule of the present invention may comprise multiple amino-terminalsand carboxy-terminals. For example, the “amino-terminus” of acell-targeting molecule may be defined by the first amino acid residueof a polypeptide chain representing the amino-terminal end of thepolypeptide, which is generally characterized by a starting, amino acidresidue which does not have a peptide bond with any amino acid residueinvolving the primary amino group of the starting amino acid residue orinvolving the equivalent nitrogen for starting amino acid residues whichare members of the class of N-alkylated alpha amino acid residues.Similarly, the “carboxy-terminus” of a cell-targeting molecule may bedefined by the last amino acid residue of a polypeptide chainrepresenting the carboxyl-terminal end of the polypeptide, which isgenerally characterized by a final, amino acid residue which does nothave any amino acid residue linked by a peptide bond to the alpha-carbonof its primary carboxyl group.

For purposes of the present invention, the terms “terminus,”“amino-terminus,” or “carboxy-terminus” with regard to a polypeptideregion refers to the regional boundaries of that region, regardless ofwhether additional amino acid residues are linked by peptide bondsoutside of that region. In other words, the terminals of the polypeptideregion regardless of whether that region is fused to other peptides orpolypeptides. For example, a fusion protein comprising two proteinaceousregions, e.g., a binding region comprising a peptide or polypeptide anda Shiga toxin effector polypeptide, may have a Shiga toxin effectorpolypeptide region with a carboxy-terminus ending at amino acid residue251 of the Shiga toxin effector polypeptide region despite a peptidebond involving residue 251 to an amino acid residue at position 252representing the beginning of another proteinaceous region, e.g., thebinding region. In this example, the carboxy-terminus of the Shiga toxineffector polypeptide region refers to residue 251, which is not aterminus of the fusion protein but rather represents an internal,regional boundary. Thus, for polypeptide regions, the terms “terminus,”“amino-terminus,” and “carboxy-terminus” are used to refer to theboundaries of polypeptide regions, whether the boundary is a physicallyterminus or an internal, position embedded within a larger polypeptidechain.

For purposes of the present invention, the phrase “carboxy-terminusregion of a Shiga toxin A1 fragment” refers to a polypeptide regionderived from a naturally occurring Shiga toxin A1 fragment, the regionbeginning with a hydrophobic residue (e.g., V236 of StxA-A1 and SLT-1A1,and V235 of SLT-2A1) that is followed by a hydrophobic residue and theregion ending with the furin-cleavage site conserved among Shiga toxinA1 fragment polypeptides and ending at the junction between the A1fragment and the A2 fragment in native, Shiga toxin A Subunits. Forpurposes of the present invention, the carboxy-terminal region of aShiga toxin A1 fragment includes a peptidic region derived from thecarboxy-terminus of a Shiga toxin A1 fragment polypeptide, such as,e.g., a peptidic region comprising or consisting essentially of thecarboxy-terminus of a Shiga toxin A1 fragment. Non-limiting examples ofpeptidic regions derived from the carboxy-terminus of a Shiga toxin A1fragment include the amino acid residue sequences natively positionedfrom position 236 to position 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, or 251 in Stx1A (SEQ ID NO:2) or SLT-1A (SEQ IDNO:1); and from position 235 to position 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, or 250 in SLT-2A (SEQ ID NO:3).

For purposes of the present invention, the phrase “proximal to thecarboxy-terminus of an A1 fragment polypeptide” with regard to a linkedmolecular moiety and/or binding region refers to being within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues from the amino acidresidue defining the last residue of the Shiga toxin A1 fragmentpolypeptide.

For purposes of the present invention, the phrase “sterically covers thecarboxy-terminus of the A1 fragment-derived region” includes anymolecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) linked and/or fused to an amino acidresidue in the carboxy-terminus of the A1 fragment-derived region, suchas, e.g., the amino acid residue derived from the amino acid residuenatively positioned at any one of positions 236 to 251 in Stx1A (SEQ IDNO:2) or SLT-1A (SEQ ID NO:1) or from 235 to 250 in SLT-2A (SEQ IDNO:3). For purposes of the present invention, the phrase “stericallycovers the carboxy-terminus of the A1 fragment-derived region” alsoincludes any molecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) linked and/or fused to an amino acidresidue in the carboxy-terminus of the A1 fragment-derived region, suchas, e.g., the amino acid residue carboxy-terminal to the last amino acidA1 fragment-derived region and/or the Shiga toxin effector polypeptide.For purposes of the present invention, the phrase “sterically covers thecarboxy-terminus of the A1 fragment-derived region” also includes anymolecular moiety of a size of 4.5 kDa or greater (e.g., animmunoglobulin-type binding region) physically preventing cellularrecognition of the carboxy-terminus of the A1 fragment-derived region,such as, e.g. recognition by the ERAD machinery of a eukaryotic cell.

For purposes of the present invention, a binding region, such as, e.g.,an immunoglobulin-type binding region, that comprises a polypeptidecomprising at least forty amino acids and that is linked (e.g., fused)to the carboxy-terminus of the Shiga toxin effector polypeptide regioncomprising an A1 fragment-derived region is a molecular moiety which is“sterically covering the carboxy-terminus of the A1 fragment-derivedregion.”

For purposes of the present invention, a binding region, such as, e.g.,an immunoglobulin-type binding region, that comprises a polypeptidecomprising at least forty amino acids and that is linked (e.g., fused)to the carboxy-terminus of the Shiga toxin effector polypeptide regioncomprising an A1 fragment-derived region is a molecular moiety“encumbering the carboxy-terminus of the A1 fragment-derived region.”

For purposes of the present invention, the term “A1 fragment of a memberof the Shiga toxin family” refers to the remaining amino-terminalfragment of a Shiga toxin A Subunit after proteolysis by furin at thefurin-cleavage site conserved among Shiga toxin A Subunits andpositioned between the A1 fragment and the A2 fragment in wild-typeShiga toxin A Subunits.

For purposes of the claimed invention, the phrase “furin-cleavage motifat the carboxy-terminus of the A1 fragment region” refers to a specific,furin-cleavage motif conserved among Shiga toxin A Subunits and bridgingthe junction between the A1 fragment and the A2 fragment in naturallyoccurring, Shiga toxin A Subunits.

For purposes of the present invention, the phrase “furin-cleavage siteproximal to the carboxy-terminus of the A1 fragment region” refers toany identifiable, furin-cleavage site having an amino acid residuewithin a distance of less than 1, 2, 3, 4, 5, 6, 7, or more amino acidresidues of the amino acid residue defining the last amino acid residuein the A1 fragment region or A1 fragment derived region, including afurin-cleavage motif located carboxy-terminal of an A1 fragment regionor A1 fragment derived region, such as, e.g., at a position proximal tothe linkage of the A1 fragment-derived region to another component ofthe molecule, such as, e.g., a molecular moiety of a cell-targetingmolecule of the present invention.

For purposes of the present invention, the phrase “disruptedfurin-cleavage motif” refers to (i) a specific furin-cleavage motif asdescribed herein in Section I-B and (ii) which comprises a mutationand/or truncation that can confer a molecule with a reduction infurin-cleavage as compared to a reference molecule, such as, e.g., areduction in furin-cleavage reproducibly observed to be 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or less (including 100% for nocleavage) than the furin-cleavage of a reference molecule observed inthe same assay under the same conditions. The percentage offurin-cleavage as compared to a reference molecule can be expressed as aratio of cleaved:uncleaved material of the molecule of interest dividedby the cleaved:uncleaved material of the reference molecule (seeExamples, supra). Non-limiting examples of suitable reference moleculesinclude certain molecules comprising a wild-type Shiga toxinfurin-cleavage motif and/or furin-cleavage site as described herein inSection I-B, Section IV-B, and/or the Examples) and/or molecules used asreference molecules in the Examples below.

For purposes of the present invention, the phrase “furin-cleavageresistant” means a molecule or specific polypeptide region thereofexhibits reproducibly less furin cleavage than (i) the carboxy-terminusof a Shiga toxin A1 fragment in a wild-type Shiga toxin A Subunit or(ii) the carboxy-terminus of the Shiga toxin A1 fragment derived regionof construct wherein the naturally occurring furin-cleavage sitenatively positioned at the junction between the A1 and A2 fragments isnot disrupted; as assayed by any available means to the skilled worker,including by using a method described herein.

For purposes of the present invention, the phrase “active enzymaticdomain derived form an A Subunit of a member of the Shiga toxin family”refers to a polypeptide structure having the ability to inhibit proteinsynthesis via catalytic inactivation of a ribosome based on a Shigatoxin enzymatic activity. The ability of a molecular structure toexhibit inhibitory activity of protein synthesis and/or catalyticinactivation of a ribosome may be observed using various assays known tothe skilled worker, such as, e.g., in vitro assays involving RNAtranslation assays in the absence of living cells or in vivo assaysinvolving the ribosomes of living cells. For example, using assays knownto the skilled worker, the enzymatic activity of a molecule based on aShiga toxin enzymatic activity may be assessed directly by observingN-glycosidase activity toward ribosomal RNA (rRNA), such as, e.g., aribosome nicking assay, and/or indirectly by observing inhibition ofribosome function, RNA translation, and/or protein synthesis.

As used herein with respect to a Shiga toxin effector polypeptide, a“combination” describes a Shiga toxin effector polypeptide comprisingtwo or more sub-regions wherein each sub-region comprises at least oneof the following: (1) a disruption in an endogenous epitope or epitoperegion; (2) an embedded, heterologous, T-cell epitope-peptide; (3) aninserted, heterologous, T-cell epitope-peptide; and (4) a disruptedfurin-cleavage motif at the carboxy-terminus of an A1 fragment region.

Introduction

The present invention provides various, combination, Shiga toxineffector polypeptides and cell-targeting molecules comprising the same.Certain embodiments of the Shiga toxin effector polypeptides of thepresent invention combine structural elements resulting in two or moreproperties in a single molecule, such as, e.g, the ability to 1) exhibitreduced antigenicity and/or immunogenicity as compared to molecularvariants lacking that particular combination of elements, 2) exhibitreduced protease-cleavage as compared to molecular variants lacking thatparticular combination of elements, 3) exhibit reduced non-specifictoxicity to a multicellular organism at certain dosages as compared tomolecular variants lacking that particular combination of elements, 4)deliver an embedded or inserted T-cell epitope to the MHC class I systema cell for cell-surface presentation, and/or 5) exhibit potentcytotoxicity. The Shiga toxin effector polypeptides of the presentinvention may serve as scaffolds to create various cell-targetingmolecules, such as, e.g., cell-targeted, cytotoxic, therapeuticmolecules; cell-targeted, nontoxic, delivery vehicles; andcell-targeted, diagnostic molecules.

I. The General Structures of the Shiga Toxin Effector Polypeptides ofthe Invention

The Shiga toxin effector polypeptides and cell-targeting molecules ofthe present invention comprise at least one, Shiga toxin effectorpolypeptide derived from wild-type Shiga toxin A Subunits but compriseone or more structural modifications, such as, e.g., a mutation like atruncation and/or amino acid residue substitution(s). For certainembodiments, the present invention involves the engineering of improved,Shiga toxin A Subunit effector polypeptides comprising the combinationof two or more of the following Shiga toxin effector polypeptidesub-regions: (1) a de-immunized sub-region, (2) a protease-cleavageresistant sub-region near the carboxy-terminus of a Shiga toxin A1fragment region, and (3) a T-cell epitope-peptide embedded or insertedsub-region.

A Shiga toxin effector polypeptide is a polypeptide derived from a Shigatoxin A Subunit member of the Shiga toxin family that is capable ofexhibiting one or more Shiga toxin functions (see e.g., Cheung M et al.,Mol Cancer 9: 28 (2010); WO 2014/164693; WO 2015/113005; WO 2015/113007;WO 2015/138452; WO 2015/191764). Shiga toxin functions include, e.g.,increasing cellular internalization, directing subcellular routing froman endosomal compartment to the cytosol, avoiding intracellulardegradation, catalytically inactivating ribosomes, and effectuatingcytostatic and/or cytotoxic effects.

The Shiga toxin family of protein toxins is composed of variousnaturally occurring toxins which are structurally and functionallyrelated, e.g., Shiga toxin, Shiga-like toxin 1, and Shiga-like toxin 2(Johannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)). Holotoxinmembers of the Shiga toxin family contain targeting domains thatpreferentially bind a specific glycosphingolipid present on the surfaceof some host cells and an enzymatic domain capable of permanentlyinactivating ribosomes once inside a cell (Johannes L, Römer W, Nat RevMicrobiol 8: 105-16 (2010)). Members of the Shiga toxin family share thesame overall structure and mechanism of action (Engedal N et al.,Microbial Biotech 4: 32-46 (2011)). For example, Stx, SLT-1 and SLT-2display indistinguishable enzymatic activity in cell free systems (HeadS et al., J Biol Chem 266: 3617-21 (1991); Tesh V et al., Infect Immun61: 3392-402 (1993); Brigotti M et al., Toxicon 35:1431-1437 (1997)).

The Shiga toxin family encompasses true Shiga toxin (Stx) isolated fromS. dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stx1 orSLT-1 or Slt-I) isolated from serotypes of enterohemorrhagic E. coli,and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2) isolated fromserotypes of enterohemorrhagic E. coli. SLT1 differs by only one aminoacid residue from Stx, and both have been referred to as Verocytotoxinsor Verotoxins (VTs) (O'Brien A, Curr Top Microbiol Immunol 180: 65-94(1992)). Although SLT1 and SLT2 variants are only about 53-60% similarto each other at the primary amino acid sequence level, they sharemechanisms of enzymatic activity and cytotoxicity common to the membersof the Shiga toxin family (Johannes L, Römer W, Nat Rev Microbiol 8:105-16 (2010)). Over 39 different Shiga toxins have been described, suchas the defined subtypes Stx1a, Stx1c, Stx1d, and Stx2a-g (Scheutz F etal., J Clin Microbiol 50: 2951-63 (2012)). Members of the Shiga toxinfamily are not naturally restricted to any bacterial species becauseShiga-toxin-encoding genes can spread among bacterial species viahorizontal gene transfer (Strauch E et al., Infect Immun 69: 7588-95(2001); Bielaszewska M et al., Appl Environ Micrbiol 73: 3144-50 (2007);Zhaxybayeva O, Doolittle W, Curr Biol 21: R242-6 (2011)). As an exampleof interspecies transfer, a Shiga toxin was discovered in a strain of A.haemolyticus isolated from a patient (Grotiuz G et al., J Clin Microbiol44: 3838-41 (2006)). Once a Shiga toxin encoding polynucleotide enters anew subspecies or species, the Shiga toxin amino acid sequence ispresumed to be capable of developing slight sequence variations due togenetic drift and/or selective pressure while still maintaining amechanism of cytotoxicity common to members of the Shiga toxin family(see Scheutz F et al., J Clin Microbiol 50: 2951-63 (2012)).

A. De-Immunized, Shiga Toxin a Subunit Effector Polypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention is de-immunized, such as, e.g., as compared to awild-type Shiga toxin, wild-type Shiga toxin polypeptide, and/or Shigatoxin effector polypeptide comprising only wild-type polypeptidesequences. The de-immunized, Shiga toxin effector polypeptides of thepresent invention each comprise a disruption of at least one, putative,endogenous, epitope region in order to reduce the antigenic and/orimmunogenic potential of the Shiga toxin effector polypeptide afteradministration of the polypeptide to a chordate. A Shiga toxin effectorpolypeptide and/or Shiga toxin A Subunit polypeptide, whether naturallyoccurring or not, can be de-immunized by a method described herein,described in WO 2015/113005 and/or WO 2015/113007, and/or known to theskilled worker, wherein the resulting molecule retains one or more Shigatoxin A Subunit functions.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises a disruption of an endogenous epitope orepitope region, such as, e.g., a B-cell and/or CD4+ T-cell epitope. Incertain embodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption of at least one, endogenous, epitoperegion described herein, wherein the disruption reduces the antigenicand/or immunogenic potential of the Shiga toxin effector polypeptideafter administration of the polypeptide to a chordate, and wherein theShiga toxin effector polypeptide is capable of exhibiting one or moreShiga toxin A Subunit functions, such as, e.g., a significant level ofShiga toxin cytotoxicity.

The term “disrupted” or “disruption” as used herein with regard to anepitope region refers to the deletion of at least one amino acid residuein an epitope region, inversion of two or more amino acid residues whereat least one of the inverted amino acid residues is in an epitoperegion, insertion of at least one amino acid into an epitope region, anda substitution of at least one amino acid residue in an epitope region.An epitope region disruption by mutation includes amino acidsubstitutions with non-standard amino acids and/or non-natural aminoacids. Epitope regions may alternatively be disrupted by mutationscomprising the modification of an amino acid by the addition of acovalently-linked chemical structure which masks at least one amino acidin an epitope region, see, e.g. PEGylation (see Zhang C et al., BioDrugs26: 209-15 (2012), small molecule adjuvants (Flower D, Expert Opin DrugDiscov 7: 807-17 (2012), and site-specific albumination (Lim S et al., JControl Release 207-93 (2015)).

Certain epitope regions and disruptions are indicated herein byreference to specific amino acid positions of native Shiga toxin ASubunits provided in the Sequence Listing, noting that naturallyoccurring Shiga toxin A Subunits may comprise precursor forms containingsignal sequences of about 22 amino acids at their amino-terminals whichare removed to produce mature Shiga toxin A Subunits and arerecognizable to the skilled worker. Further, certain epitope regiondisruptions are indicated herein by reference to specific amino acids(e.g. S for a serine residue) natively present at specific positionswithin native Shiga toxin A Subunits (e.g. S33 for the serine residue atposition 33 from the amino-terminus) followed by the amino acid withwhich that residue has been substituted in the particular mutation underdiscussion (e.g. S33I represents the amino acid substitution ofisoleucine for serine at amino acid residue 33 from the amino-terminus).

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises a disruption of at leastone epitope region provided herein (see e.g. Tables 1-7 and/or 12). Incertain embodiments, the de-immunized, Shiga toxin effector polypeptideof the present invention comprises a disruption of at least one epitoperegion described in WO 2015/113005 or WO 2015/113007.

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention comprises or consists essentiallyof a full-length Shiga toxin A Subunit (e.g. SLT-1A (SEQ ID NO:1), StxA(SEQ ID NO:2), or SLT-2A (SEQ ID NO:3)) comprising at least onedisruption of the amino acid sequence selected from the group ofnatively positioned amino acids consisting of: 1-15 of SEQ ID NO:1 orSEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ IDNO:1 or SEQ ID NO:2; 39-48 of SEQ ID NO:1 or SEQ ID NO:2; 42-48 of SEQID NO:3; 53-66 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2;179-191 of SEQ ID NO: 3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQID NO:2; 210-218 of SEQ ID NO:3; 240-258 of SEQ ID NO:3; 243-257 of SEQID NO:1 or SEQ ID NO:2; 254-268 of SEQ ID NO:1 or SEQ ID NO:2; 262-278of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO:1 orSEQ ID NO:2, or the equivalent position in a Shiga toxin A Subunitpolypeptide, conserved Shiga toxin effector polypeptide sub-region,and/or non-native, Shiga toxin effector polypeptide sequence.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises or consists essentially of a truncated Shigatoxin A Subunit.

Truncations of Shiga toxin A Subunits might result in the deletion of anentire epitope region(s) without affecting Shiga toxin effectorfunction(s). The smallest, Shiga toxin A Subunit fragment shown toexhibit significant enzymatic activity was a polypeptide composed ofresidues 75-247 of StxA (A1-Jaufy A et al., Infect Immun 62: 956-60(1994)). Truncating the carboxy-terminus of SLT-1A, StxA, or SLT-2A toamino acids 1-251 removes two predicted B-cell epitope regions, twopredicted CD4 positive (CD4+) T-cell epitopes, and a predicted,discontinuous, B-cell epitope. Truncating the amino-terminus of SLT-1A,StxA, or SLT-2A to 75-293 removes at least three, predicted, B-cellepitope regions and three predicted CD4+ T-cell epitopes. Truncatingboth amino- and carboxy-terminals of SLT-1A, StxA, or SLT-2A to 75-251deletes at least five, predicted, B-cell epitope regions; four,putative, CD4+ T-cell epitopes; and one, predicted, discontinuous,B-cell epitope.

In certain embodiments, a Shiga toxin effector polypeptide of theinvention may comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one mutation, e.g.deletion, insertion, inversion, or substitution, in a provided epitoperegion. In certain further embodiments, the polypeptides comprise adisruption which comprises a deletion of at least one amino acid withinthe epitope region. In certain further embodiments, the polypeptidescomprise a disruption which comprises an insertion of at least one aminoacid within the epitope region. In certain further embodiments, thepolypeptides comprise a disruption which comprises an inversion of aminoacids, wherein at least one inverted amino acid is within the epitoperegion. In certain further embodiments, the polypeptides comprise adisruption which comprises a mutation, such as an amino acidsubstitution to a non-standard amino acid or an amino acid with achemically modified side chain. Numerous examples of single amino acidsubstitutions are provided in the Examples below.

In certain embodiments, the Shiga toxin effector polypeptides of theinvention may comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with one or more mutations as comparedto the native sequence which comprises at least one amino acidsubstitution selected from the group consisting of: A, G, V, L, I, P, C,M, F, S, D, N, Q, H, and K. In certain further embodiments, thepolypeptide may comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with a single mutation as compared tothe native sequence wherein the substitution is selected from the groupconsisting of: D to A, D to G, D to V, D to L, D to I, D to F, D to S, Dto Q, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, Eto N, E to D, E to M, E to R, G to A, H to A, H to G, H to V, H to L, Hto I, H to F, H to M, K to A, K to G, K to V, K to L, K to I, K to M, Kto H, L to A, L to G, N to A, N to G, N to V, N to L, N to I, N to F, Pto A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, Rto M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, Sto I, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, Tto M, T to S, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, and Y toM.

In certain embodiments, the Shiga toxin effector polypeptides of theinvention comprise or consist essentially of a full-length or truncatedShiga toxin A Subunit with one or more mutations as compared to thenative amino acid residue sequence which comprises at least one aminoacid substitution of an immunogenic residue and/or within an epitoperegion, wherein at least one substitution occurs at the nativelypositioned group of amino acids selected from the group consisting of: 1of SEQ ID NO:1 or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ IDNO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ IDNO:2; 46 of SEQ ID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ IDNO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1or SEQ ID NO:2; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 orSEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1 or SEQID NO:2; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1 or SEQ IDNO:2; 57 of SEQ ID NO:1 or SEQ ID NO:2; 58 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 ofSEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ IDNO:1 or SEQ ID NO:2; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 96of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1 or SEQ IDNO:2; 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQ IDNO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;141 of SEQ ID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;184 of SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO:3; 185 of SEQ ID NO:1 orSEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ IDNO:1 or SEQ ID NO:2; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ IDNO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ IDNO:3; 248 of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ IDNO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQID NO:2.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one substitution of animmunogenic residue and/or within an epitope region, wherein at leastone amino acid substitution is to a non-conservative amino acid (see,e.g., Table C, infra) relative to a natively occurring amino acidpositioned at one of the following native positions: 1 of SEQ ID NO:1 orSEQ ID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ IDNO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1 orSEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ IDNO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1or SEQ ID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 orSEQ ID NO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1 or SEQID NO:2; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 84 ofSEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1 or SEQ ID NO: 2; 94 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 96 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO: 3; 104 of SEQ ID NO:1 or SEQ ID NO:2; 105 of SEQ ID NO:1or SEQ ID NO:2; 107 of SEQ ID NO:1 or SEQ ID NO:2; 108 of SEQ ID NO:1 orSEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1 or SEQ ID NO:2; 112 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO: 3; 141 of SEQ ID NO:1 or SEQ ID NO:2;147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 orSEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 198 of SEQ IDNO:1 or SEQ ID NO:2; 204 of SEQ ID NO: 3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ IDNO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2;and 286 of SEQ ID NO:1 or SEQ ID NO:2..

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one amino acidsubstitution selected from the group consisting of: K1to A, G, V, L, I,F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S,and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, M, and S;T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11 toA, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, and S; S33 to A,G, V, L, I, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L;S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to Aand P; D47 to A, G, V, L, I, F, S, and Q; N48 to A, G, V, L, and M; L49to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A,G, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P;157 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F,S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M,and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 toA, G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H;V88 to A and G; 188 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F,M, and S; S107 to A, G, V, L, I, F, and M; L108 to A, G, and M; S109 toA, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A;D111 to A, G, V, L, I, F, S, and Q; S112 to A, G, V, L, I, F, and M;D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G; R179 toA, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S;T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q;D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, and V; S186 to A, G,V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, andH; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L, I, F, S, and Q;D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S,K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V,and S; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M;R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F,M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; C262 toA, G, V, and S; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286to A, G, V, L, I, F, M, and S.

In certain further embodiments, the Shiga toxin effector polypeptides ofthe invention comprise or consist essentially of a full-length ortruncated Shiga toxin A Subunit with at least one of the following aminoacid substitutions K1A, K1M, T4I, D6R, S81, T8V, T9I, S9I, K11A, K11H,T12K, S33I, S33C, S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G,N48V, N48F, L49A, F50T, A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V,R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E601, E60T, E60R,E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I, T104N, A105L, T107P,L108M, S109V, T109V, G110A, D111T, S112V, D141A, G147A, V154A, R179A,T180G, T1811, D183A, D183G, D184A, D184A, D184F, L185V, L185D, S186A,S186F, G187A, G187T, R188A, R188L, S189A, D198A, R204A, R205A, C242S,S2471, Y247A, R248A, R250A, R251A, or D264A, G264A, T286A, and/or T286I.These epitope disrupting substitutions may be combined to form ade-immunized, Shiga toxin effector polypeptide with multiplesubstitutions per epitope region and/or multiple epitope regionsdisrupted while still retaining Shiga toxin effector function. Forexample, substitutions at the natively positioned K1A, K1M, T4I, D6R,S81, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N, G44L, S45V,S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A,D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V,D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A,D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A, D111T,S112V, D141A, G147A, V154A, R179A, T180G, T1811, D183A, D183G, D184A,D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L,S189A, D198A, R204A, R205A, C242S, S2471, Y247A, R248A, R250A, R251A, orD264A, G264A, T286A, and/or T286I may be combined, where possible, withsubstitutions at the natively positioned residues K1A, K1M, T4I, D6R,S81, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N, G44L, S45V,S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T, A51V, D53A,D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V,D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A, R84A, V88A,D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A, D111T,S112V, D141A, G147A, V154A, R179A, T180G, T1811, D183A, D183G, D184A,D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A, R188L,S189A, D198A, R204A, R205A, C242S, S2471, Y247A, R248A, R250A, R251A, orD264A, G264A, T286A, and/or T286I to create de-immunized, Shiga toxineffector polypeptides of the invention.

Any of the de-immunized, Shiga toxin effector polypeptide sub-regionsand/or epitope disrupting mutations described herein may be used aloneor in combination with each individual embodiment of the presentinvention, including methods of the present invention.

B. Protease-Cleavage Resistant, Shiga Toxin a Subunit EffectorPolypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derived regionhaving a carboxy-terminus and (2) a disrupted furin-cleavage motif atthe carboxy-terminus of the Shiga toxin A1 fragment region. Improvingthe stability of connections between the Shiga toxin component and othercomponents of cell-targeting molecules, e.g., cell-targeting bindingregions, can improve their toxicity profiles after administration toorganisms by reducing non-specific toxicities caused by the breakdown ofthe connection and loss of cell-targeting, such as, e.g., as a result ofproteolysis.

Shiga toxin A Subunits of members of the Shiga toxin family comprise aconserved, furin-cleavage site at the carboxy-terminal of their A1fragment regions important for Shiga toxin function. Furin-cleavage sitemotifs and furin-cleavage sites can be identified by the skilled workerusing standard techniques and/or by using the information herein.

The model of Shiga toxin cytotoxicity is that intracellular proteolyticprocessing of Shiga toxin A Subunits by furin in intoxicated cells isessential for 1) liberation of the A1 fragment from the rest of theShiga holotoxin, 2) escape of the A1 fragment from the endoplasmicreticulum by exposing a hydrophobic domain in the carboxy-terminus ofthe A1 fragment, and 3) enzymatic activation of the A1 fragment (seeJohannes L, Römer W, Nat Rev Microbiol 8: 105-16 (2010)). The efficientliberation of the Shiga toxin A1 fragment from the A2 fragment and therest of the components of the Shiga holotoxin in the endoplasmicreticulum of intoxicated cells is essential for efficient intracellularrouting to the cytosol, maximal enzymatic activity, efficient ribosomeinactivation, and achieving optimal cytotoxicity, i.e. comparable to awild-type Shiga toxin (see e.g. WO 2015/191764 and references therein).

During Shiga toxin intoxication, the A Subunit is proteolyticallycleaved by furin at the carboxy bond of a conserved arginine residue(e.g. the arginine residue at position 251 in StxA and SLT-1A and thearginine residue at position 250 in Stx2A and SLT-2A). Furin cleavage ofShiga toxin A Subunits occurs in endosomal and/or Golgi compartments.Furin is a specialized serine endoprotease which is expressed by a widevariety of cell types, in all human tissues examined, and by most animalcells. Furin cleaves polypeptides comprising accessible motifs oftencentered on the minimal, dibasic, consensus motif R-x-(R/K/x)-R. The ASubunits of members of the Shiga toxin family comprise a conserved,surface-exposed, extended loop structure (e.g. 242-261 in StxA andSLT-1A, and 241-260 in SLT-2) with a conserved S-R/Y-x-x-R motif whichis cleaved by furin. The surface exposed, extended loop structurepositioned at amino acid residues 242-261 in StxA is required forfurin-induced cleavage of StxA, including features flanking the minimal,furin-cleavage motif R-x-x-R.

Furin-cleavage motifs and furin-cleavage sites in Shiga toxin A Subunitsand Shiga toxin effector polypeptides can be identified by the skilledworker using standard methods and/or by using the information herein.Furin cleaves the minimal, consensus motif R-x-x-R (Schalken J et al., JClin Invest 80: 1545-9 (1987); Bresnahan P et al., J Cell Biol 111:2851-9 (1990); Hatsuzawa K et al., J Biol Chem 265: 22075-8 (1990); WiseR et al., Proc Natl Acad Sci USA 87: 9378-82 (1990); Molloy S et al., JBiol Chem 267: 16396-402 (1992)). Consistent with this, many furininhibitors comprise peptides comprising the motif R-x-x-R. An example ofa synthetic inhibitor of furin is a molecule comprising the peptideR-V-K-R (SEQ ID NO:537) (Henrich S et al., Nat Struct Biol 10: 520-6(2003)). In general, a peptide or protein comprising a surfaceaccessible, dibasic amino acid motif with two positively charged, aminoacids separated by two amino acid residues may be predicted to besensitive to furin-cleavage with cleavage occurring at the carboxy bondof the last basic amino acid in the motif.

Consensus motifs in substrates cleaved by furin have been identifiedwith some degree of specificity. A furin-cleavage site motif has beendescribed that comprises a region of twenty, continuous, amino acidresidues, which can be labeled P14 through P6′ (Tian S et al., Int J MolSci 12: 1060-5 (2011)) using the nomenclature described in Schechter I,Berger, A, Biochem Biophys Res Commun 32: 898-902 (1968). According tothis nomenclature, the furin-cleavage site is at the carboxy bond of theamino acid residue designated P1, and the amino acid residues of thefurin-cleavage motif are numbered P2, P3, P4, etc., in the directiongoing toward the amino-terminus from this reference P1 residue. Theamino acid residues of the motif going toward the carboxy-terminus fromthe P1 reference residue are numbered with the prime notation P2′, P3′,P4′, etc. Using this nomenclature, the P6 to P2′ region delineates thecore substrate of the furin cleavage motif which is bound by theenzymatic domain of furin. The two flanking regions P14 to P7 and P3′ toP6′ are often rich in polar, amino acid residues to increase theaccessibility to the core furin cleavage site located between them.

A general, furin-cleavage site is often described by the consensus motifR-x-x-R which corresponds to P4-P3-P2-P1; where “R” represents anarginine residue (see Table A, supra), a dash “-” represents a peptidebond, and a lowercase “x” represents any amino acid residue. However,other residues and positions may help to further define furin-cleavagemotifs. A slightly more refined furin-cleavage site, consensus motif isoften reported as the consensus motif R-x-[K/R]-R (where a forward slash“/” means “or” and divides alternative amino acid residues at the sameposition), which corresponds to P4-P3-P2-P1, because it was observedthat furin has a strong preference for cleaving substrates containingthis motif.

In addition to the minimal, furin-cleavage site R-x-x-R, a larger,furin-cleavage motif has been described with certain amino acid residuepreferences at certain positions. By comparing various known furinsubstrates, certain physicochemical properties have been characterizedfor the amino acids residues in a 20 amino acid residue long,furin-cleavage site motif. The P6 to P2′ region of the furin-cleavagemotif delineates the core furin-cleavage site which physically interactswith the enzymatic domain of furin. The two flanking regions P14 to P7and P3′ to P6′ are often hydrophilic being rich in polar, amino acidresidues to increase the surface accessibility of the corefurin-cleavage site located between them.

In general, the furin-cleavage motif region from position P5 to P1 tendsto comprise amino acid residues with a positive charge and/or highisoelectric points. In particular, the P1 position, which marks theposition of furin proteolysis, is generally occupied by an arginine butother positively charged, amino acid residues may occur in thisposition. Positions P2 and P3 tend to be occupied by flexible, aminoacid residues, and in particular P2 tends to be occupied by arginine,lysine, or sometimes by very small and flexible amino acid residues likeglycine. The P4 position tends to be occupied by positively charged,amino acid residues in furin substrates. However, if the P4 position isoccupied by an aliphatic, amino acid residue, then the lack of apositively charged, functional group can be compensated for by apositively charged residue located at position(s) P5 and/or P6.Positions P1′ and P2′ are commonly occupied by aliphatic and/orhydrophobic amino acid residues, with the P1′ position most commonlybeing occupied by a serine.

The two, hydrophilic, flanking regions tend to be occupied by amino acidresidues which are polar, hydrophilic, and have smaller amino acidfunctional groups; however, in certain verified furin substrates, theflanking regions do not contain any hydrophilic, amino acid residues(see Tian S, Biochem Insights 2: 9-20 (2009)).

The twenty amino acid residue, furin-cleavage motif and furin-cleavagesite found in native, Shiga toxin A Subunits at the junction between theShiga toxin A1 fragment and A2 fragment is well characterized in certainShiga toxins. For example in StxA (SEQ ID NO:2) and SLT-1A (SEQ IDNO:1), this furin-cleavage motif is natively positioned from L238 toF257, and in SLT-2A (SEQ ID NO:3), this furin-cleavage motif is nativelypositioned from V237 to Q256. Based on amino acid homology, experiment,and/or furin-cleavage assays described herein, the skilled worker canidentify furin-cleavage motifs in other native, Shiga toxin A Subunitsor Shiga toxin effector polypeptides, where the motifs are actualfurin-cleavage motifs or are predicted to result in the production of A1and A2 fragments after furin cleavage of those molecules within aeukaryotic cell.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derivedpolypeptide having a carboxy-terminus and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment derivedpolypeptide. The carboxy-terminus of a Shiga toxin A1 fragment derivedpolypeptide may be identified by the skilled worker by using techniquesknown in the art, such as, e.g., by using protein sequence alignmentsoftware to identify (i) a furin-cleavage motif conserved with anaturally occurring Shiga toxin, (ii) a surface exposed, extended loopconserved with a naturally occurring Shiga toxin, and/or (iii) a stretchof amino acid residues which are predominantly hydrophobic (i.e. ahydrophobic “patch”) that may be recognized by the ERAD system.

A protease-cleavage resistant, Shiga toxin effector polypeptide of thepresent invention (1) may be completely lacking any furin-cleavage motifat a carboxy-terminus of its Shiga toxin A1 fragment region and/or (2)comprise a disrupted furin-cleavage motif at the carboxy-terminus of itsShiga toxin A1 fragment region and/or region derived from thecarboxy-terminus of a Shiga toxin A1 fragment. A disruption of afurin-cleavage motif include various alterations to an amino acidresidue in the furin-cleavage motif, such as, e.g., a post-translationmodification(s), an alteration of one or more atoms in an amino acidfunctional group, the addition of one or more atoms to an amino acidfunctional group, the association to a non-proteinaceous moiety(ies),and/or the linkage to an amino acid residue, peptide, polypeptide suchas resulting in a branched proteinaceous structure.

Protease-cleavage resistant, Shiga toxin effector polypeptides may becreated from a Shiga toxin effector polypeptide and/or Shiga toxin ASubunit polypeptide, whether naturally occurring or not, using a methoddescribed herein, described in WO 2015/191764, and/or known to theskilled worker, wherein the resulting molecule still retains one or moreShiga toxin A Subunit functions.

For purposes of the present invention with regard to a furin-cleavagesite or furin-cleavage motif, the term “disruption” or “disrupted”refers to an alteration from the naturally occurring furin-cleavage siteand/or furin-cleavage motif, such as, e.g., a mutation, that results ina reduction in furin-cleavage proximal to the carboxy-terminus of aShiga toxin A1 fragment region, or identifiable region derived thereof,as compared to the furin-cleavage of a wild-type Shiga toxin A Subunitor a polypeptide derived from a wild-type Shiga toxin A Subunitcomprising only wild-type polypeptide sequences. An alteration to anamino acid residue in the furin-cleavage motif includes a mutation inthe furin-cleavage motif, such as, e.g., a deletion, insertion,inversion, substitution, and/or carboxy-terminal truncation of thefurin-cleavage motif, as well as a post-translation modification, suchas, e.g., as a result of glycosylation, albumination, and the like whichinvolve conjugating or linking a molecule to the functional group of anamino acid residue. Because the furin-cleavage motif is comprised ofabout twenty, amino acid residues, in theory, alterations,modifications, mutations, deletions, insertions, and/or truncationsinvolving one or more amino acid residues of any one of these twentypositions might result in a reduction of furin-cleavage sensitivity(Tian S et al., Sci Rep 2: 261 (2012)). The disruption of afurin-cleavage site and/or furin-cleavage motif may or may not increaseresistance to cleavage by other proteases, such as, e.g., trypsin andextracellular proteases common in the vascular system of mammals. Theeffects of a given disruption to cleavage sensitivity of a givenprotease may be tested by the skilled worker using techniques known inthe art.

For purposes of the present invention, a “disrupted furin-cleavagemotif” is furin-cleavage motif comprising an alteration to one or moreamino acid residues derived from the 20 amino acid residue regionrepresenting a conserved, furin-cleavage motif found in native, Shigatoxin A Subunits at the junction between the Shiga toxin A1 fragment andA2 fragment regions and positioned such that furin cleavage of a Shigatoxin A Subunit results in the production of the A1 and A2 fragments;wherein the disrupted furin-cleavage motif exhibits reduced furincleavage in an experimentally reproducible way as compared to areference molecule comprising a wild-type, Shiga toxin A1 fragmentregion fused to a carboxy-terminal polypeptide of a size large enough tomonitor furin cleavage using the appropriate assay known to the skilledworker and/or described herein.

Examples of types of mutations which can disrupt a furin-cleavage siteand furin-cleavage motif are amino acid residue deletions, insertions,truncations, inversions, and/or substitutions, including substitutionswith non-standard amino acids and/or non-natural amino acids. Inaddition, furin-cleavage sites and furin-cleavage motifs can bedisrupted by mutations comprising the modification of an amino acid bythe addition of a covalently-linked structure which masks at least oneamino acid in the site or motif, such as, e.g., as a result ofPEGylation, the coupling of small molecule adjuvants, and/orsite-specific albumination.

If a furin-cleavage motif has been disrupted by mutation and/or thepresence of non-natural amino acid residues, certain disruptedfurin-cleavage motifs may not be easily recognizable as being related toany furin-cleavage motif, however, the carboxy-terminus of the Shigatoxin A1 fragment derived region will be recognizable and will definewhere the furin-cleavage motif would be located were it not disrupted.For example, a disrupted furin-cleavage motif may comprise less than thetwenty, amino acid residues of the furin-cleavage motif due to acarboxy-terminal truncation as compared to a Shiga toxin A Subunitand/or Shiga toxin A1 fragment.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises (1) a Shiga toxin A1 fragment derivedpolypeptide having a carboxy-terminus and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of the Shiga toxin A1 fragment polypeptideregion; wherein the Shiga toxin effector polypeptide (and anycell-targeting molecule comprising it) is more furin-cleavage resistantas compared to a reference molecule, such as, e.g., a wild-type Shigatoxin polypeptide comprising the carboxy-terminus of an A1 fragmentand/or the conserved, furin-cleavage motif between A1 and A2 fragments.For example, a reduction in furin cleavage of one molecule compared to areference molecule may be determined using an in vitro, furin-cleavageassay described in the Examples below, conducted using the sameconditions, and then performing a quantitation of the band density ofany fragments resulting from cleavage to quantitatively measure inchange in furin cleavage.

In certain embodiments, the Shiga toxin effector polypeptide is moreresistant to furin-cleavage in vitro and/or in vivo as compared to awild-type, Shiga toxin A Subunit.

In general, the protease-cleavage sensitivity of a cell-targetingmolecule of the present invention is tested by comparing it to the samemolecule having its furin-cleavage resistant, Shiga toxin effectorpolypeptide replaced with a wild-type, Shiga toxin effector polypeptidecomprising a Shiga toxin A1 fragment. In certain embodiments, themolecules of the present invention comprising a disrupted furin-cleavagemotif exhibits a reduction in in vitro furin cleavage of 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 98% or greater compared to a referencemolecule comprising a wild-type, Shiga toxin A1 fragment fused at itscarboxy-terminus to a peptide or polypeptide, such as, e.g., thereference molecule SLT-1A-WT::scFv-1 described in Example 2, below.

Several furin-cleavage motif disruptions have been described. Forexample, mutating the two conserved arginines to alanines in the minimalR-x-x-R motif completely blocked processing by furin and/or furin-likeproteases (see e.g Duda A et al., J Virology 78: 13865-70 (2004)).Because the furin-cleavage site motif is comprised of about twenty aminoacid residues, in theory, certain mutations involving one or more of anyone of these twenty, amino acid residue positions might abolish furincleavage or reduce furin cleavage efficiency (see e.g. Tian S et al.,Sci Rep 2: 261 (2012)).

In certain embodiments, the molecules of the present invention comprisea Shiga toxin effector polypeptide derived from at least one A Subunitof a member of the Shiga toxin family wherein the Shiga toxin effectorpolypeptide comprises a disruption in one or more amino acids derivedfrom the conserved, highly accessible, protease-cleavage sensitive loopof Shiga toxin A Subunits. For example, in StxA and SLT-1A, this highlyaccessible, protease-sensitive loop is natively positioned from aminoacid residues 242 to 261, and in SLT-2A, this conserved loop is nativelypositioned from amino acid residues 241 to 260. Based on polypeptidesequence homology, the skilled worker can identify this conserved,highly accessible loop structure in other Shiga toxin A Subunits.Certain mutations to the amino acid residues in this loop can reduce theaccessibility of certain amino acid residues within the loop toproteolytic cleavage and this might reduce furin-cleavage sensitivity.

In certain embodiments, a molecule of the present invention comprises aShiga toxin effector polypeptide comprising a disrupted furin-cleavagemotif comprising a mutation in the surface-exposed, protease sensitiveloop conserved among Shiga toxin A Subunits. In certain furtherembodiments, a molecule of the present invention comprises a Shiga toxineffector polypeptide comprising a disrupted furin-cleavage motifcomprising a mutation in this protease-sensitive loop of Shiga toxin ASubunits, the mutation which reduce the surface accessibility of certainamino acid residues within the loop such that furin-cleavage sensitivityis reduced.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in terms of existence, position, or functional group of oneor both of the consensus amino acid residues P1 and P4, such as, e.g.,the amino acid residues in positions 1 and 4 of the minimalfurin-cleavage motif R/Y-x-x-R. For example, mutating one or both of thetwo arginine residues in the minimal, furin consensus site R-x-x-R toalanine will disrupt a furin-cleavage motif and prevent furin-cleavageat that site. Similarly, amino acid residue substitutions of one or bothof the arginine residues in the minimal furin-cleavage motif R-x-x-R toany non-conservative amino acid residue known to the skilled worker willreduced the furin-cleavage sensitivity of the motif. In particular,amino acid residue substitutions of arginine to any non-basic amino acidresidue which lacks a positive charge, such as, e.g., A, G, P, S, T, D,E, Q, N, C, I, L, M, V, F, W, and Y, will result in a disruptedfurin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif of a Shigatoxin effector polypeptide of the present invention comprises adisruption in the spacing between the consensus amino acid residues P4and P1 in terms of the number of intervening amino acid residues beingother than two, and, thus, changing either P4 and/or P1 into a differentposition and eliminating the P4 and/or P1 designations. For example,deletions within the furin-cleavage motif of the minimal furin-cleavagesite or the core, furin-cleavage motif will reduce the furin-cleavagesensitivity of the furin-cleavage motif.

In certain embodiments, the disrupted furin-cleavage motif comprises oneor more amino acid residue substitutions, as compared to a wild-type,Shiga toxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residuesubstitutions within the minimal furin-cleavage site R/Y-x-x-R, such as,e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides, thenatively positioned amino acid residue R248 substituted with anynon-positively charged, amino acid residue and/or R251 substituted withany non-positively charged, amino acid residue; and for SLT-2A derivedShiga toxin effector polypeptides, the natively positioned amino acidresidue Y247 substituted with any non-positively charged, amino acidresidue and/or R250 substituted with any non-positively charged, aminoacid residue.

In certain embodiments, the disrupted furin-cleavage motif comprises anun-disrupted, minimal furin-cleavage site R/Y-x-x-R but insteadcomprises a disrupted flanking region, such as, e.g., amino acid residuesubstitutions in one or more amino acid residues in the furin-cleavagemotif flanking regions natively position at, e.g., 241-247 and/or252-259. In certain further embodiments, the disrupted furin cleavagemotif comprises a substitution of one or more of the amino acid residueslocated in the P1-P6 region of the furin-cleavage motif, mutating P1′ toa bulky amino acid, such as, e.g., R, W, Y, F, and H; and mutating P2′to a polar and hydrophilic amino acid residue; and substituting one ormore of the amino acid residues located in the P1′-P6′ region of thefurin-cleavage motif with one or more bulky and hydrophobic amino acidresidues

In certain embodiments, the disruption of the furin-cleavage motifcomprises a deletion, insertion, inversion, and/or mutation of at leastone amino acid residue within the furin-cleavage motif. In certainembodiments, a protease-cleavage resistant, Shiga toxin effectorpolypeptide of the present invention may comprise a disruption of theamino acid sequence natively positioned at 249-251 of the A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) or Shiga toxin (SEQ ID NO:2), or at247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NO:3) or theequivalent position in a conserved Shiga toxin effector polypeptideand/or non-native Shiga toxin effector polypeptide sequence. In certainfurther embodiments, protease-cleavage resistant, Shiga toxin effectorpolypeptides comprise a disruption which comprises a deletion of atleast one amino acid within the furin-cleavage motif. In certain furtherembodiments, protease-cleavage resistant, Shiga toxin effectorpolypeptides comprise a disruption which comprises an insertion of atleast one amino acid within the protease-cleavage motif region. Incertain further embodiments, the protease-cleavage resistant, Shigatoxin effector polypeptides comprise a disruption which comprises aninversion of amino acids, wherein at least one inverted amino acid iswithin the protease motif region. In certain further embodiments, theprotease-cleavage resistant, Shiga toxin effector polypeptides comprisea disruption which comprises a mutation, such as an amino acidsubstitution to a non-standard amino acid or an amino acid with achemically modified side chain. Examples of single amino acidsubstitutions are provided in the Examples below.

In certain embodiments of the molecules of the present invention, thedisrupted furin-cleavage motif comprises the deletion of nine, ten,eleven, or more of the carboxy-terminal amino acid residues within thefurin-cleavage motif. In these embodiments, the disrupted furin-cleavagemotif will not comprise a furin-cleavage site or a minimalfurin-cleavage motif. In other words, certain embodiments lack afurin-cleavage site at the carboxy-terminus of the A1 fragment region.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue deletion and an amino acid residuesubstitution as compared to a wild-type, Shiga toxin A Subunit. Incertain further embodiments, the disrupted furin-cleavage motifcomprises one or more amino acid residue deletions and substitutionswithin the minimal furin-cleavage site R/Y-x-x-R, such as, e.g., forStxA and SLT-1A derived Shiga toxin effector polypeptides, the nativelypositioned amino acid residue R248 substituted with any non-positivelycharged, amino acid residue and/or R251 substituted with anynon-positively charged, amino acid residue; and for SLT-2A derived Shigatoxin effector polypeptides, the natively positioned amino acid residueY247 substituted with any non-positively charged, amino acid residueand/or R250 substituted with any non-positively charged, amino acidresidue.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion and an amino acid residue substitution aswell as a carboxy-terminal truncation as compared to a wild-type, Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises one or more amino acid residue deletionsand substitutions within the minimal furin-cleavage site R/Y-x-x-R, suchas, e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides,the natively positioned amino acid residue R248 substituted with anynon-positively charged, amino acid residue and/or R251 substituted withany non-positively charged, amino acid residue; and for SLT-2A derivedShiga toxin effector polypeptides, the natively positioned amino acidresidue Y247 substituted with any non-positively charged, amino acidresidue and/or R250 substituted with any non-positively charged, aminoacid residue.

In certain further embodiments, the disrupted furin-cleavage motifcomprises both an amino acid substitution within the minimalfurin-cleavage site R/Y-x-x-R and a carboxy-terminal truncation ascompared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxAand SLT-1A derived Shiga toxin effector polypeptides, truncations endingat the natively amino acid position 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, or greater and comprising thenatively positioned amino acid residue R248 and/or R251 substituted withany non-positively charged, amino acid residue where appropriate; andfor SLT-2A derived Shiga toxin effector polypeptides, truncations endingat the natively amino acid position 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, or greater and comprisingthe natively positioned amino acid residue Y247 and/or R250 substitutedwith any non-positively charged, amino acid residue where appropriate.

In certain embodiments, the disrupted furin-cleavage motif comprises aninsertion of one or more amino acid residues as compared to a wild-type,Shiga toxin A Subunit as long as the inserted amino residue(s) does notcreate a de novo furin-cleavage site. In certain embodiments, theinsertion of one or more amino acid residues disrupts the naturalspacing between the arginine residues in the minimal, furin-cleavagesite R/Y-x-x-R, such as, e.g., StxA and SLT-1A derived polypeptidescomprising an insertion of one or more amino acid residues at 249 or 250and thus between R248 and R251; or SLT-2A derived polypeptidescomprising an insertion of one or more amino acid residues at 248 or 249and thus between Y247 and R250.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue insertion and a carboxy-terminal truncationas compared to a wild-type, Shiga toxin A Subunit. In certainembodiments, the disrupted furin-cleavage motif comprises both an aminoacid residue insertion and an amino acid residue substitution ascompared to a wild-type, Shiga toxin A Subunit. In certain embodiments,the disrupted furin-cleavage motif comprises both an amino acid residueinsertion and an amino acid residue deletion as compared to a wild-type,Shiga toxin A Subunit.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion, an amino acid residue insertion, and anamino acid residue substitution as compared to a wild-type, Shiga toxinA Subunit.

In certain embodiments, the disrupted furin-cleavage motif comprises anamino acid residue deletion, insertion, substitution, andcarboxy-terminal truncation as compared to a wild-type, Shiga toxin ASubunit.

In certain embodiments, the Shiga toxin effector polypeptide comprisinga disrupted furin-cleavage motif is directly fused by a peptide bond toa molecular moiety comprising an amino acid, peptide, and/or polypeptidewherein the fused structure involves a single, continuous polypeptide.In these fusion embodiments, the amino acid sequence following thedisrupted furin-cleavage motif should not create a de novo,furin-cleavage site at the fusion junction.

Any of the above protease-cleavage resistant, Shiga toxin effectorpolypeptide sub-regions and/or disrupted furin-cleavage motifs may beused alone or in combination with each individual embodiment of thepresent invention, including methods of the present invention.

C. T-Cell Hyper-Immunized, Shiga Toxin a Subunit Effector Polypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises an embedded or inserted epitope-peptide. Incertain further embodiments, the epitope-peptide is a heterologous,T-cell epitope-peptide, such as, e.g., an epitope consideredheterologous to Shiga toxin A Subunits. In certain further embodiments,the epitope-peptide is a CD8+ T-cell epitope. In certain furtherembodiments, the CD8+ T-cell epitope-peptide has a binding affinity to aMHC class I molecule characterized by a dissociation constant (K_(D)) of10⁻⁴ molar or less and/or the resulting MHC class I-epitope-peptidecomplex has a binding affinity to a T-cell receptor (TCR) characterizedby a dissociation constant (K_(D)) of 10⁻⁴ molar or less.

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises an embedded or inserted, heterologous,T-cell epitope, such as, e.g., a human CD8+ T-cell epitope. In certainfurther embodiments, the heterologous, T-cell epitope is embedded orinserted so as to disrupt an endogenous epitope or epitope region (e.g.a B-cell epitope and/or CD4+ T-cell epitope) identifiable in a naturallyoccurring Shiga toxin polypeptide or parental Shiga toxin effectorpolypeptide from which the Shiga toxin effector polypeptide of thepresent invention is derived.

For certain embodiments of the present invention, the Shiga toxineffector polypeptide (and any cell-targeting molecule comprising it) isCD8+ T-cell hyper-immunized, such as, e.g., as compared to a wild-typeShiga toxin polypeptide. The CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides of the present invention each comprise an embeddedor inserted T-cell epitope-peptide. Hyper-immunized, Shiga toxineffector polypeptides can be created from Shiga toxin effectorpolypeptides and/or Shiga toxin A Subunit polypeptides, whethernaturally occurring or not, using a method described herein, describedin WO 2015/113007, and/or known to the skilled worker, wherein theresulting molecule still retains one or more Shiga toxin A Subunitfunctions.

For purposes of the claimed invention, a T-cell epitope is a molecularstructure which is comprised by an antigenic peptide and can berepresented by a linear, amino acid sequence. Commonly, T-cell epitopesare peptides of sizes of eight to eleven amino acid residues (TownsendA, Bodmer H, Annu Rev Immunol 7: 601-24 (1989)); however, certain T-cellepitope-peptides have lengths that are smaller than eight or larger thaneleven amino acids long (see e.g. Livingstone A, Fathman C, Annu RevImmunol 5: 477-501 (1987); Green K et al., Eur J Immunol 34: 2510-9(2004)). In certain embodiments, the embedded or inserted epitope is atleast seven amino acid residues in length. In certain embodiments, theembedded or inserted epitope is bound by a TCR with a binding affinitycharacterized by a K_(D) less than 10 mM (e.g. 1-100 pM) as calculatedusing the formula in Stone J et al., Immunology 126: 165-76 (2009).However, it should be noted that the binding affinity within a givenrange between the MHC-epitope and TCR may not correlate withantigenicity and/or immunogenicity (see e.g. A1-Ramadi B et al., JImmunol 155: 662-73 (1995)), such as due to factors like MHC-peptide-TCRcomplex stability, MHC-peptide density and MHC-independent functions ofTCR cofactors such as CD8 (Baker B et al., Immunity 13: 475-84 (2000);Hornell T et al., J Immunol 170: 4506-14 (2003); Woolridge L et al., JImmunol 171: 6650-60 (2003)).

A heterologous, T-cell epitope is an epitope not already present in awild-type Shiga toxin A Subunit; a naturally occurring Shiga toxin ASubunit; and/or a parental, Shiga toxin effector polypeptide used as asource polypeptide for modification by a method described herein,described in WO 2015/113007, and/or known to the skilled worker.

A heterologous, T-cell epitope-peptide may be incorporated into a sourcepolypeptide via numerous methods known to the skilled worker, including,e.g., the processes of creating one or more amino acid substitutionswithin the source polypeptide, fusing one or more amino acids to thesource polypeptide, inserting one or more amino acids into the sourcepolypeptide, linking a peptide to the source polypeptide, and/or acombination of the aforementioned processes. The result of such a methodis the creation of a modified variant of the source polypeptide whichcomprises one or more embedded or inserted, heterologous, T-cellepitope-peptides.

T-cell epitopes may be chosen or derived from a number of sourcemolecules for use in the present invention. T-cell epitopes may becreated or derived from various naturally occurring proteins. T-cellepitopes may be created or derived from various naturally occurringproteins foreign to mammals, such as, e.g., proteins of microorganisms.T-cell epitopes may be created or derived from mutated human proteinsand/or human proteins aberrantly expressed by malignant human cells.T-cell epitopes may be synthetically created or derived from syntheticmolecules (see e.g., Carbone F et al., J Exp Med 167: 1767-9 (1988); DelVal M et al., J Virol 65: 3641-6 (1991); Appella E et al., Biomed PeptProteins Nucleic Acids 1: 177-84 (1995); Perez S et al., Cancer 116:2071-80 (2010)).

Although any T-cell epitope-peptide is contemplated as being used as aheterologous, T-cell epitope of the present invention, certain epitopesmay be selected based on desirable properties. One objective of thepresent invention is to create CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides for administration to vertebrates, meaning thatthe heterologous, T-cell epitope is highly immunogenic and can elicitrobust immune responses in vivo when displayed complexed with a NMCclass I molecule on the surface of a cell. In certain embodiments, theShiga toxin effector polypeptide of the present invention comprises oneor more, embedded or inserted, heterologous, T-cell epitopes which areCD8+ T-cell epitopes. A Shiga toxin effector polypeptide of the presentinvention that comprises a heterologous, CD8+ T-cell epitope isconsidered a CD8+ T-cell hyper-immunized, Shiga toxin effectorpolypeptide.

T-cell epitope components of the present invention may be chosen orderived from a number of source molecules already known to be capable ofeliciting a vertebrate immune response. T-cell epitopes may be derivedfrom various naturally occurring proteins foreign to vertebrates, suchas, e.g., proteins of pathogenic microorganisms and non-self, cancerantigens. In particular, infectious microorganisms may contain numerousproteins with known antigenic and/or immunogenic properties. Further,infectious microorganisms may contain numerous proteins with knownantigenic and/or immunogenic sub-regions or epitopes.

For example, the proteins of intracellular pathogens with mammalianhosts are sources for T-cell epitopes. There are numerous intracellularpathogens, such as viruses, bacteria, fungi, and single-cell eukaryotes,with well-studied antigenic proteins or peptides. T-cell epitopes can beselected or identified from human viruses or other intracellularpathogens, such as, e.g., bacteria like mycobacterium, fungi liketoxoplasmae, and protists like trypanosomes.

For example, there are many immunogenic, viral peptide components ofviral proteins from viruses that are infectious to humans. Numerous,human T-cell epitopes have been mapped to peptides within proteins frominfluenza A viruses, such as peptides in the proteins HA glycoproteinsFE17, 5139/1, CH65, C05, hemagglutin 1 (HA1), hemagglutinin 2 (HA2),nonstructural protein 1 and 2 (NS1 and NS 2), matrix protein 1 and 2 (M1and M2), nucleoprotein (NP), neuraminidase (NA)), and many of thesepeptides have been shown to elicit human immune responses, such as byusing ex vivo assay. Similarly, numerous, human T-cell epitopes havebeen mapped to peptide components of proteins from humancytomegaloviruses (HCMV), such as peptides in the proteins pp65 (UL83),UL128-131, immediate-early 1 (IE-1; UL123), glycoprotein B, tegumentproteins, and many of these peptides have been shown to elicit humanimmune responses, such as by using ex vivo assays.

Another example is there are many immunogenic, cancer antigens inhumans. The CD8+ T-cell epitopes of cancer and/or tumor cell antigenscan be identified by the skilled worker using techniques known in theart, such as, e.g., differential genomics, differential proteomics,immunoproteomics, prediction then validation, and genetic approacheslike reverse-genetic transfection (see e.g., Admon A et al., Mol CellProteomics 2: 388-98 (2003); Purcell A, Gorman J, Mol Cell Proteomics 3:193-208 (2004); Comber J, Philip R, Ther Adv Vaccines 2: 77-89 (2014)).There are many antigenic and/or immunogenic T-cell epitopes alreadyidentified or predicted to occur in human cancer and/or tumor cells. Forexample, T-cell epitopes have been predicted in human proteins commonlymutated or overexpressed in neoplastic cells, such as, e.g., ALK, CEA,N-acetylglucosaminyl-transferase V (GnT-V), HCA587, HER-2/neu, MAGE,Melan-A/MART-1, MUC-1, p53, and TRAG-3 (see e.g., van der Bruggen P etal., Science 254: 1643-7 (1991); Kawakami Y et al., J Exp Med 180:347-52 (1994); Fisk B et al., J Exp Med 181: 2109-17 (1995); Guilloux Yet al., J Exp Med 183: 1173 (1996); Skipper J et al., J Exp Med 183: 527(1996); Brossart P et al., 93: 4309-17 (1999); Kawashima I et al.,Cancer Res 59: 431-5 (1999); Papadopoulos K et al., Clin Cancer Res 5:2089-93 (1999); Zhu B et al., Clin Cancer Res 9: 1850-7 (2003); Li B etal., Clin Exp Immunol 140: 310-9 (2005); Ait-Tahar K et al., Int JCancer 118: 688-95 (2006); Akiyama Y et al., Cancer Immunol Immunother61: 2311-9 (2012)). In addition, synthetic variants of T-cell epitopesfrom human cancer cells have been created (see e.g., Lazoura E,Apostolopoulos V, Curr Med Chem 12: 629-39 (2005); Douat-Casassus C etal., J Med Chem 50: 1598-609 (2007)).

While any T-cell epitope may be used in the polypeptides and moleculesof the present invention, certain T-cell epitopes may be preferred basedon their known and/or empirically determined characteristics. Forexample, in many species, the MHC alleles in its genome encode multipleMHC-I molecular variants. Because MHC class I protein polymorphisms canaffect antigen-MHC class I complex recognition by CD8+ T-cells, T-cellepitopes may be chosen for use in the present invention based onknowledge about certain MHC class I polymorphisms and/or the ability ofcertain antigen-MHC class I complexes to be recognized by T-cells havingdifferent genotypes.

There are well-defined peptide-epitopes that are known to beimmunogenic, MHC class I restricted, and/or matched with a specifichuman leukocyte antigen (HLA) variant(s). For applications in humans orinvolving human target cells, HLA-class I-restricted epitopes can beselected or identified by the skilled worker using standard techniquesknown in the art. The ability of peptides to bind to human MHC class Imolecules can be used to predict the immunogenic potential of putativeT-cell epitopes. The ability of peptides to bind to human MHC class Imolecules can be scored using software tools. T-cell epitopes may bechosen for use as a heterologous, T-cell epitope component of thepresent invention based on the peptide selectivity of the HLA variantsencoded by the alleles more prevalent in certain human populations. Forexample, the human population is polymorphic for the alpha chain of MHCclass I molecules due to the varied alleles of the HLA genes fromindividual to individual. In certain T-cell epitopes may be moreefficiently presented by a specific HLA molecule, such as, e.g., thecommonly occurring HLA variants encoded by the HLA-A allele groupsHLA-A2 and HLA-A3.

When choosing T-cell epitopes for use as a heterologous, T-cell epitopecomponent of the present invention, multiple factors may be consideredthat can influence epitope generation and transport to receptive MHCclass I molecules, such as, e.g., the presence and epitope specificityof the following factors in the target cell: proteasome, ERAAP/ERAP1,tapasin, and TAPs.

When choosing T-cell epitopes for use as a heterologous, T-cell epitopecomponent of the present invention, epitope may be selected which bestmatch the MHC class I molecules present in the cell-type or cellpopulations to be targeted. Different MHC class I molecules exhibitpreferential binding to particular peptide sequences, and particularpeptide-MHC class I variant complexes are specifically recognized by thet-cell receptors (TCRs) of effector T-cells. The skilled worker can useknowledge about MHC class I molecule specificities and TCR specificitiesto optimize the selection of heterologous, T-cell epitopes used in thepresent invention.

In addition, multiple, immunogenic, T-cell epitopes for MHC class Ipresentation may be embedded in the same Shiga toxin effectorpolypeptide of the present invention, such as, e.g., for use in thetargeted delivery of a plurality of T-cell epitopes simultaneously. Anexample of a cell-targeting molecule of the present invention comprisingmultiple, CD8+ T-cell epitopes is SEQ ID NO:26.

Any of the protease-cleavage resistant, Shiga toxin effector polypeptidesub-regions and/or disrupted furin-cleavage motifs described herein maybe used alone or in combination with each individual embodiment of thepresent invention, including methods of the present invention.

II. The General Structures of the Cell-Targeting Molecules of theInvention

The Shiga toxin effector polypeptides of the present invention providerobust and powerful scaffolds for engineering novel, cell-targetingmolecules. The associated of cell-targeting binding regions with Shigatoxin effector polypeptides of the present invention enables theengineering of therapeutic and diagnostic molecules with desirablecharacteristics, such as, e.g., de-immunization, potent cytotoxicity,efficient intracellular routing, T-cell hyper-immunization, molecularstability, and in vivo tolerability at high dosages.

The present invention provides various cell-targeting molecules, eachcomprising (1) a cell-targeting, binding region and (2) a Shiga toxineffector polypeptide of the present invention. The Shiga toxin effectorpolypeptides of the present invention may be associated with and/orcoupled to various, diverse, cell-targeting components (e.g. a molecularmoiety and/or agent) to create cell-targeting molecules of the presentinvention. A cell-targeting molecule of the present invention comprises(1) a binding region capable of specifically binding an extracellularpart of a target biomolecule and (2) a Shiga toxin effector polypeptideregion comprising a Shiga toxin effector polypeptide of the presentinvention.

The Shiga toxin effector polypeptides of the present invention may belinked to one or more cell-targeting, binding regions that mediatecell-targeting via binding specificity to extracellular parts of targetbiomolecules, such as, e.g., a target biomolecule physically coupled toa cellular surface of a cell. One non-limiting example of acell-targeting molecule of the present invention is a Shiga toxineffector polypeptide of the present invention fused to a proteinaceous,cell-targeting, binding region, such as, e.g., an immunoglobulin-typebinding region.

A. Binding Regions

In certain embodiments, a binding region of a cell-targeting molecule ofthe present invention is a cell-targeting component, such as, e.g., adomain, molecular moiety, or agent, capable of binding specifically toan extracellular part of a target biomolecule (e.g. an extracellulartarget biomolecule) with high affinity. There are numerous types ofbinding regions known to skilled worker or which may be discovered bythe skilled worker using techniques known in the art. For example, anycell-targeting component that exhibits the requisite bindingcharacteristics described herein may be used as the binding region incertain embodiments of the cell-targeting molecules of the presentinvention.

An extracellular part of a target biomolecule refers to a portion of itsstructure exposed to the extracellular environment when the molecule isphysically coupled to a cell, such as, e.g., when the target biomoleculeis expressed at a cellular surface by the cell. In this context, exposedto the extracellular environment means that part of the targetbiomolecule is accessible by, e.g., an antibody or at least a bindingmoiety smaller than an antibody such as a single-domain antibody domain,a nanobody, a heavy-chain antibody domain derived from camelids orcartilaginous fishes, a single-chain variable fragment, or any number ofengineered alternative scaffolds to immunoglobulins (see below). Theexposure to the extracellular environment of or accessibility to a partof target biomolecule physically coupled to a cell may be empiricallydetermined by the skilled worker using methods well known in the art.

A binding region of a cell-targeting molecule of the present inventionmay be, e.g., a ligand, peptide, immunoglobulin-type binding region,monoclonal antibody, engineered antibody derivative, or engineeredalternative to antibodies.

In certain embodiments, the binding region of a cell-targeting moleculeof the present invention is a proteinaceous moiety capable of bindingspecifically to an extracellular part of target biomolecule with highaffinity. A binding region of a cell-targeting molecule of the presentinvention may comprise one or more various peptidic or polypeptidemoieties, such as randomly generated peptide sequences, naturallyoccurring ligands or derivatives thereof, immunoglobulin deriveddomains, synthetically engineered scaffolds as alternatives toimmunoglobulin domains, and the like (see e.g., WO 2005/092917; WO2007/033497; Cheung M et al., Mol Cancer 9: 28 (2010); US 2013/0196928;WO 2014/164693; WO 2015/113005; WO 2015/113007; WO 2015/138452; WO2015/191764). In certain embodiments, a cell-targeting molecule of thepresent invention comprises a binding region comprising one or morepolypeptides capable of selectively and specifically binding anextracellular target biomolecule.

There are numerous binding regions known in the art that are useful fortargeting molecules to specific cell-types via their bindingcharacteristics, such as certain ligands, monoclonal antibodies,engineered antibody derivatives, and engineered alternatives toantibodies.

According to one specific but non-limiting aspect, the binding region ofa cell-targeting molecule of the present invention comprises a naturallyoccurring ligand or derivative thereof that retains bindingfunctionality to an extracellular target biomolecule, commonly a cellsurface receptor. For example, various cytokines, growth factors, andhormones known in the art may be used to target the cell-targetingmolecule of the present invention to the cell-surface of specific celltypes expressing a cognate cytokine receptor, growth factor receptor, orhormone receptor. Certain non-limiting examples of ligands include(alternative names are indicated in parentheses) agiogenin, B-cellactivating factors (BAFFs, APRIL), colony stimulating factors (CSFs),epidermal growth factors (EGFs), fibroblast growth factors (FGFs),vascular endothelial growth factors (VEGFs), insulin-like growth factors(IGFs), interferons, interleukins (such as IL-2, IL-6, and IL-23), nervegrowth factors (NGFs), platelet derived growth factors, transforminggrowth factors (TGFs), and tumor necrosis factors (TNFs).

According to certain other embodiments of the cell-targeting moleculesof the present invention, the binding region comprises a syntheticligand capable of binding an extracellular target biomolecule (see e.g.Liang S et al., J Mol Med 84: 764-73 (2006); Ahmed S et al., Anal Chem82: 7533-41 (2010); Kaur K et al., Methods Mol Biol 1248: 239-47(2015)).

In certain embodiments, the binding region comprises a peptidomimetic,such as, e.g., an AApeptide, gamma-AApeptide, and/or sulfono-7-AApeptide(see e.g., Pilsl L, Reiser O, Amino Acids 41: 709-18 (2011); Akram O etal., Mol Cancer Res 12: 967-78 (2014); Wu H et al., Chemistry 21: 2501-7(2015); Teng P et al., Chemistry 2016 Mar. 4)).

According to one specific, but non-limiting aspect, the binding regionmay comprise an immunoglobulin-type binding region. The term“immunoglobulin-type binding region” as used herein refers to apolypeptide region capable of binding one or more target biomolecules,such as an antigen or epitope. Binding regions may be functionallydefined by their ability to bind to target molecules.Immunoglobulin-type binding regions are commonly derived from antibodyor antibody-like structures; however, alternative scaffolds from othersources are contemplated within the scope of the term.

Immunoglobulin (Ig) proteins have a structural domain known as an Igdomain. Ig domains range in length from about 70-110 amino acid residuesand possess a characteristic Ig-fold, in which typically 7 to 9antiparallel beta strands arrange into two beta sheets which form asandwich-like structure. The Ig fold is stabilized by hydrophobic aminoacid interactions on inner surfaces of the sandwich and highly conserveddisulfide bonds between cysteine residues in the strands. Ig domains maybe variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgIor I-set). Some Ig domains may be associated with a complementaritydetermining region (CDR), also called a “complementary determiningregion,” which is important for the specificity of antibodies binding totheir epitopes. Ig-like domains are also found in non-immunoglobulinproteins and are classified on that basis as members of the Igsuperfamily of proteins. The HUGO Gene Nomenclature Committee (HGNC)provides a list of members of the Ig-like domain containing family.

An immunoglobulin-type binding region may be a polypeptide sequence ofan antibody or antigen-binding fragment thereof wherein the amino acidsequence has been varied from that of a native antibody or an Ig-likedomain of a non-immunoglobulin protein, for example by molecularengineering or selection by library screening. Because of the relevanceof recombinant DNA techniques and in vitro library screening in thegeneration of immunoglobulin-type binding regions, antibodies can beredesigned to obtain desired characteristics, such as smaller size, cellentry, or other improvements for in vivo and/or therapeuticapplications. The possible variations are many and may range from thechanging of just one amino acid to the complete redesign of, forexample, a variable region. Typically, changes in the variable regionwill be made in order to improve the antigen-binding characteristics,improve variable region stability, or reduce the potential forimmunogenic responses.

There are numerous immunoglobulin-type binding regions contemplated ascomponents of the present invention. In certain embodiments, theimmunoglobulin-type binding region is derived from an immunoglobulinbinding region, such as an antibody paratope capable of binding anextracellular target biomolecule. In certain other embodiments, theimmunoglobulin-type binding region comprises an engineered polypeptidenot derived from any immunoglobulin domain but which functions like animmunoglobulin binding region by providing high-affinity binding to anextracellular target biomolecule. This engineered polypeptide mayoptionally include polypeptide scaffolds comprising or consistingessentially of complementary determining regions from immunoglobulins asdescribed herein.

There are also numerous binding regions in the prior art that are usefulfor targeting polypeptides to specific cell-types via theirhigh-affinity binding characteristics. In certain embodiments, thebinding region of the cell-targeting molecule of the present inventionis selected from the group which includes autonomous V_(H) domains,single-domain antibody domains (sdAbs), heavy-chain antibody domainsderived from camelids (V_(H)H fragments or V_(H) domain fragments),heavy-chain antibody domains derived from camelid V_(H)H fragments orV_(H) domain fragments, heavy-chain antibody domains derived fromcartilaginous fishes, immunoglobulin new antigen receptors (IgNARs),V_(NAR) fragments, single-chain variable (scFv) fragments, nanobodies,Fd fragments consisting of the heavy chain and C_(H)i domains, singlechain Fv-C_(H)3 minibodies, dimeric CH2 domain fragments (CH2D), Fcantigen binding domains (Fcabs), isolated complementary determiningregion 3 (CDR3) fragments, constrained framework region 3, CDR3,framework region 4 (FR3-CDR3-FR4) polypeptides, small modularimmunopharmaceutical (SMIP) domains, scFv-Fc fusions, multimerizing scFvfragments (diabodies, triabodies, tetrabodies), disulfide stabilizedantibody variable (Fv) fragments, disulfide stabilized antigen-binding(Fab) fragments consisting of the V_(L), V_(H), CL and C_(H)i domains,bivalent nanobodies, bivalent minibodies, bivalent F(ab′)₂ fragments(Fab dimers), bispecific tandem V_(H)H fragments, bispecific tandem scFvfragments, bispecific nanobodies, bispecific minibodies, and anygenetically manipulated counterparts of the foregoing that retain itsparatope and binding function (see Ward E et al., Nature 341: 544-6(1989); Davies J, Riechmann L, Biotechnology (NY) 13: 475-9 (1995);Reiter Y et al., Mol Biol 290: 685-98 (1999); Riechmann L, MuyldermansS, J Immunol Methods 231: 25-38 (1999); Tanha J et al., J ImmunolMethods 263: 97-109 (2002); Vranken W et al., Biochemistry 41: 8570-9(2002); Jespers L et al., J Mol Biol 337: 893-903 (2004); Jespers L etal., Nat Biotechnol 22: 1161-5 (2004); To R et al., J Biol Chem 280:41395-403 (2005); Saerens D et al., Curr Opin Pharmacol 8: 600-8 (2008);Dimitrov D, MAbs 1: 26-8 (2009); Weiner L, Cell 148: 1081-4 (2012);Ahmad Z et al., Clin Dev Immunol 2012: 980250 (2012)).

There are a variety of binding regions comprising polypeptides derivedfrom the constant regions of immunoglobulins, such as, e.g., engineereddimeric Fc domains, monomeric Fcs (mFcs), scFv-Fcs, V_(H)H-Fcs, CH2domains, monomeric CH3s domains (mC_(H)3s), synthetically reprogrammedimmunoglobulin domains, and/or hybrid fusions of immunoglobulin domainswith ligands (Hofer T et al., Proc Natl Acad Sci U.S.A 105: 12451-6(2008); Xiao J et al., J Am Chem Soc 131: 13616-13618 (2009); Xiao X etal., Biochem Biophys Res Commun 387: 387-92 (2009); Wozniak-Knopp G etal., Protein Eng Des Sel 23 289-97 (2010); Gong R et al., PLoS ONE 7:e42288 (2012); Wozniak-Knopp G et al., PLoS ONE 7: e30083 (2012); Ying Tet al., J Biol Chem 287: 19399-408 (2012); Ying T et al., J Biol Chem288: 25154-64 (2013); Chiang M et al., J Am Chem Soc 136: 3370-3 (2014);Rader C, Trends Biotechnol 32: 186-97 (2014); Ying T et al., BiochimicaBiophys Acta 1844: 1977-82 (2014)).

In accordance with certain other embodiments, the binding regioncomprises an engineered, alternative scaffold to immunoglobulin domains.Engineered alternative scaffolds are known in the art which exhibitsimilar functional characteristics to immunoglobulin-derived structures,such as high-affinity and specific binding of target biomolecules, andmay provide improved characteristics to certain immunoglobulin domains,such as, e.g., greater stability or reduced immunogenicity. Generally,alternative scaffolds to immunoglobulins are less than 20 kilodaltons,consist of a single polypeptide chain, lack cysteine residues, andexhibit relatively high thermodynamic stability.

For certain embodiments of the cell-targeting molecules of the presentinvention, the binding region comprises an alternative scaffold selectedfrom the group which includes autonomous V_(H) domains, single-domainantibody domains (sdAbs), heavy-chain antibody domains derived fromcamelids (V_(H)H fragments or V_(H) domain fragments), heavy-chainantibody domains derived from camelid V_(H)H fragments or V_(H) domainfragments, heavy-chain antibody domains derived from cartilaginousfishes, immunoglobulin new antigen receptors (IgNARs), V_(NAR)fragments, single-chain variable (scFv) fragments, nanobodies, Fdfragments consisting of the heavy chain and C_(H)i domains, permutatedFvs (pFv), single chain Fv-C_(H)3 minibodies, dimeric CH2 domainfragments (CH2D), Fe antigen binding domains (Fcabs), isolatedcomplementary determining region 3 (CDR3) fragments, constrainedframework region 3, CDR3, framework region 4 (FR3-CDR3-FR4)polypeptides, small modular immunopharmaceutical (SMIP) domains, scFv-Fcfusions, multimerizing scFv fragments (diabodies, triabodies,tetrabodies), disulfide stabilized antibody variable (Fv) fragments,disulfide stabilized antigen-binding (Fab) fragments consisting of theV_(L), V_(H), C_(L) and C_(H)i domains, bivalent nanobodies, bivalentminibodies, bivalent F(ab′)₂ fragments (Fab dimers), bispecific tandemV_(H)H fragments, bispecific tandem scFv fragments, bispecificnanobodies, bispecific minibodies, and any genetically manipulatedcounterparts of the foregoing that retains its binding functionality(Wörn A, Plückthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., ChemBiol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel 17: 455-62(2004); Binz H et al., Nat Biotechnol 23: 1257-68 (2005); Hey T et al.,Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, NatBiotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17:653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007);Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules16: 2467-85 (2011); Alfarano P et al., Protein Sci 21: 1298-314 (2012);Madhurantakam C et al., Protein Sci 21: 1015-28 (2012); Varadamsetty Get al., J Mol Biol 424: 68-87 (2012); Reichen C et al., J Struct Biol185: 147-62 (2014)).

For example, numerous alternative scaffolds have been identified whichbind to the extracellular receptor HER2 (see e.g. Wikman M et al.,Protein Eng Des Sel 17: 455-62 (2004); Orlova A et al. Cancer Res 66:4339-8 (2006); Ahlgren S et al., Bioconjug Chem 19: 235-43 (2008);Feldwisch J et al., J Mol Biol 398: 232-47 (2010); U.S. Pat. Nos.5,578,482; 5,856,110; 5,869,445; 5,985,553; 6,333,169; 6,987,088;7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185; 7,449,480;7,560,111; 7,674,460; 7,815,906; 7,879,325; 7,884,194; 7,993,650;8,241,630; 8,349,585; 8,389,227; 8,501,909; 8,512,967; 8,652,474; andU.S. patent application 2011/0059090). In addition to alternativeantibody formats, antibody-like binding abilities may be conferred bynon-proteinaceous compounds, such as, e.g., oligomers, RNA molecules,DNA molecules, carbohydrates, and glycocalyxcalixarenes (see e.g.Sansone F, Casnati A, Chem Soc Rev 42: 4623-39 (2013)) or partiallyproteinaceous compounds, such as, e.g., phenol-formaldehyde cyclicoligomers coupled with peptides and calixarene-peptide compositions (seee.g. U.S. Pat. No. 5,770,380).

Any of the above binding region structures may be used as a component ofa molecule of the present invention as long as the binding regioncomponent has a dissociation constant of 10⁻⁵ to 10⁻¹² moles per liter,preferably less than 200 nanomolar (nM), towards an extracellular targetbiomolecule.

In certain embodiments, the cell-targeting molecules of the presentinvention comprise a Shiga toxin effector polypeptide of the presentinvention linked and/or fused to a binding region capable ofspecifically binding an extracellular part of a target biomolecule or anextracellular target biomolecule. Extracellular target biomolecules maybe selected based on numerous criteria, such as a criterion describedherein.

B. Extracellular Target Biomolecules Bound by the Binding Regions

In certain embodiments, the binding region of a cell-targeting moleculesof the present invention comprises a proteinaceous region capable ofbinding specifically to an extracellular part of a target biomolecule oran extracellular target biomolecule, preferably which is physicallycoupled to the surface of a cell type of interest, such as, e.g., acancer cell, tumor cell, plasma cell, infected cell, or host cellharboring an intracellular pathogen. Target biomolecules bound by thebinding region of a cell-targeting molecule of the present invention mayinclude biomarkers over-proportionately or exclusively present on cancercells, immune cells, and/or cells infected with intracellular pathogens,such as, e.g., viruses, bacteria, fungi, prions, or protozoans.

The term “target biomolecule” refers to a biological molecule, commonlya proteinaceous molecule or a protein modified by post-translationalmodifications, such as glycosylation, that is bound by a binding regionof a cell-targeting molecule of the present invention resulting in thetargeting of the cell-targeting molecule to a specific cell, cell-type,and/or location within a multicellular organism.

For purposes of the present invention, the term “extracellular” withregard to a target biomolecule refers to a biomolecule that has at leasta portion of its structure exposed to the extracellular environment. Theexposure to the extracellular environment of or accessibility to a partof target biomolecule coupled to a cell may be empirically determined bythe skilled worker using methods well known in the art. Non-limitingexamples of extracellular target biomolecules include cell membranecomponents, transmembrane spanning proteins, cell membrane-anchoredbiomolecules, cell-surface-bound biomolecules, and secretedbiomolecules.

With regard to the present invention, the phrase “physically coupled”when used to describe a target biomolecule means covalent and/ornon-covalent intermolecular interactions couple the target biomolecule,or a portion thereof, to the outside of a cell, such as a plurality ofnon-covalent interactions between the target biomolecule and the cellwhere the energy of each single interaction is on the order of at leastabout 1-5 kiloCalories (e.g., electrostatic bonds, hydrogen bonds, ionicbonds, Van der Walls interactions, hydrophobic forces, etc.). Allintegral membrane proteins can be found physically coupled to a cellmembrane, as well as peripheral membrane proteins. For example, anextracellular target biomolecule might comprise a transmembrane spanningregion, a lipid anchor, a glycolipid anchor, and/or be non-covalentlyassociated (e.g. via non-specific hydrophobic interactions and/or lipidbinding interactions) with a factor comprising any one of the foregoing.

Extracellular parts of target biomolecules may include various epitopes,including unmodified polypeptides, polypeptides modified by the additionof biochemical functional groups, and glycolipids (see e.g. U.S. Pat.No. 5,091,178; EP2431743).

The binding regions of the cell-targeting molecules of the presentinvention may be designed or selected based on numerous criteria, suchas the cell-type specific expression of their target biomolecules, thephysical localization of their target biomolecules with regard tospecific cell types, and/or the properties of their target biomolecules.For example, certain cell-targeting molecules of the present inventioncomprise binding regions capable of binding cell-surface targetbiomolecules that are expressed at a cellular surface exclusively byonly one cell-type of a species or only one cell-type within amulticellular organism. It is desirable, but not necessary, that anextracellular target biomolecule be intrinsically internalized or bereadily forced to internalize upon interacting with a cell-targetingmolecule of the present invention.

Among certain embodiments of the cell-targeting molecules of the presentinvention, the binding region is derived from an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to a surfaceantigen on the cell surface of a cancer or tumor cell, where the antigenis restricted in expression to cancer or tumor cells (see Glokler J etal., Molecules 15: 2478-90 (2010); Liu Y et al., Lab Chip 9: 1033-6(2009). In accordance with other embodiments, the binding region isselected for specific and high-affinity binding to a surface antigen onthe cell surface of a cancer cell, where the antigen is over-expressedor preferentially expressed by cancer cells as compared to non-cancercells. Some representative target biomolecules include, but are notlimited to, the following enumerated targets associated with cancersand/or specific immune cell types.

Many immunoglobulin-type binding regions that bind with high affinity toextracellular epitopes associated with cancer cells are known to theskilled worker, such as binding regions that bind any one of thefollowing target biomolecules: annexin A1, B3 melanoma antigen, B4melanoma antigen, CD2, CD3, CD4, CD19, CD20 (B-lymphocyte antigenprotein CD20), CD22, CD25 (interleukin-2 receptor TL2R), CD30 (TNFRSF8),CD37, CD38 (cyclic ADP ribose hydrolase), CD40, CD44 (hyaluronanreceptor), ITGAV (CD51), CD56, CD66, CD70, CD71 (transferrin receptor),CD73, CD74 (HLA-DR antigens-associated invariant chain), CD79, CD98,endoglin (END, CD105), CD106 (VCAM-1), CD138, chemokine receptor type 4(CDCR-4, fusin, CD184), CD200, insulin-like growth factor 1 receptor(CD221), mucin1 (MUC1, CD227, CA6, CanAg), basal cell adhesion molecule(B-CAM, CD239), CD248 (endosialin, TEM1), tumor necrosis factor receptor10b (TNFRSF10B, CD262), tumor necrosis factor receptor 13B (TNFRSF13B,TACI, CD276), vascular endothelial growth factor receptor 2 (KDR,CD309), epithelial cell adhesion molecule (EpCAM, CD326), humanepidermal growth factor receptor 2 (HER2, Neu, ErbB2, CD340), cancerantigen 15-3 (CA15-3), cancer antigen 19-9 (CA 19-9), cancer antigen 125(CA125, MUC16), CA242, carcinoembryonic antigen-related cell adhesionmolecules (e.g. CEACAM3 (CD66d) and CEACAM5), carcinoembryonic antigenprotein (CEA), choline transporter-like protein 4 (SLC44A4), chondroitinsulfate proteoglycan 4 (CSP4, MCSP, NG2), CTLA4, delta-like proteins(e.g. DLL3, DLL4), ectonucleotide pyrophosphatase/phosphodiesteraseproteins (e.g. ENPP3), endothelin receptors (ETBRs), epidermal growthfactor receptor (EGFR, ErbB1), folate receptors (FOLRs, e.g. FRα), G-28,ganglioside GD2, ganglioside GD3, HLA-Dr10, HLA-DRB, human epidermalgrowth factor receptor 1 (HER1), HER3/ErbB-3, Ephrin type-B receptor 2(EphB2), epithelial cell adhesion molecule (EpCAM), fibroblastactivation protein (FAP/seprase), guanylyl cyclase c (GCC), insulin-likegrowth factor 1 receptor (IGF1R), interleukin 2 receptor (IL-2R),interleukin 6 receptor (IL-6R), integrins alpha-V beta-3 (α_(v)β₃),integrins alpha-V beta-5 (αvβ5), integrins alpha-5 beta-1 (α₅β₃), L6,zinc transporter (LIV-1), MPG, melanoma-associated antigen 1 protein(MAGE-1), melanoma-associated antigen 3 (MAGE-3), mesothelin (MSLN),metalloreductase STEAP1, MPG, MS4A, NaPi2b, nectins (e.g. nectin-4),p21, p97, polio virus receptor-like 4 (PVRL4),protease-activated-receptors (such as PAR1), prostate-specific membraneantigen proteins (PSMAs), SLIT and NTRK-like proteins (e.g. SLITRK6),Thomas-Friedenreich antigen, transmembrane glycoprotein (GPNMB),trophoblast glycoproteins (TPGB, 5T4, WAIF1), and tumor-associatedcalcium signal transducers (TACSTDs, e.g. Trop-2, EGP-1, etc.) (see e.g.Lui B et al., Cancer Res 64: 704-10 (2004); Novellino L et al., CancerImmunol Immunother 54: 187-207 (2005); Bagley R et al., Int J Oncol 34:619-27 (2009); Gerber H et al., mAbs 1: 247-53 (2009); Beck A et al.,Nat Rev Immunol 10: 345-52 (2010); Andersen J et al., J Biol Chem 287:22927-37 (2012); Nolan-Stevaux O et al., PLoS One 7: e50920 (2012); RustS et al., Mol Cancer 12: 11 (2013)). This list of target biomolecules isintended to be non-limiting. It will be appreciated by the skilledworker that any desired target biomolecule associated with a cancer cellor other desired cell type may be used to design or select a bindingregion which may be suitable for use as a component of a cell-targetingmolecule of the present invention.

Examples of other target biomolecules which are strongly associated withcancer cells and are bound with high-affinity by a knownimmunoglobulin-type binding region include BAGE proteins (B melanomaantigens), basal cell adhesion molecules (BCAMs or Lutheran blood groupglycoproteins), bladder tumor antigen (BTA), cancer-testis antigenNY-ESO-1, cancer-testis antigen LAGE proteins, CD19 (B-lymphocyteantigen protein CD19), CD21 (complement receptor-2 or complement 3dreceptor), CD26 (dipeptidyl peptidase-4, DPP4, or adenosine deaminasecomplexing protein 2), CD33 (sialic acid-binding immunoglobulin-typelectin-3), CD52 (CAMPATH-1 antigen), CD56, CS1 (SLAM family number 7 orSLAMF7), cell surface A33 antigen protein (gpA33), Epstein-Barr virusantigen proteins, GAGE/PAGE proteins (melanoma associated cancer/testisantigens), hepatocyte growth factor receptor (HGFR or c-Met), MAGEproteins, melanoma antigen recognized by T-cells 1 protein(MART-1/MelanA, MARTI), mucins, Preferentially Expressed Antigen ofMelanoma (PRAME) proteins, prostate specific antigen protein (PSA),prostate stem cell antigen protein (PSCA), Receptor for AdvancedGlycation Endroducts (RAGE), tumor-associated glycoprotein 72 (TAG-72),vascular endothelial growth factor receptors (VEGFRs), and Wilms' tumorantigen.

Examples of other target biomolecules which are strongly associated withcancer cells are carbonic anhydrase IX (CA9/CAIX), claudin proteins(CLDN3, CLDN4), ephrin type-A receptor 3 (EphA3), folate bindingproteins (FBP), ganglioside GM2, insulin-like growth factor receptors,integrins (such as CD11a-c), receptor activator of nuclear factor kappaB (RANK), receptor tyrosine-protein kinase erB-3, tumor necrosis factorreceptor 10A (TRAIL-R1/DR4), tumor necrosis factor receptor 10B(TRAIL-R2), tenascin C, and CD64 (FcγTRI) (see Hough C et al., CancerRes 60: 6281-7 (2000); Thepen T et al., Nat Biotechnol 18: 48-51 (2000);Pastan I et al., Nat Rev Cancer 6: 559-65 (2006); Pastan, Annu Rev Med58: 221-37 (2007); Fitzgerald D et al., Cancer Res 71: 6300-9 (2011);Scott A et al., Cancer Immun 12: 14-22 (2012)). This list of targetbiomolecules is intended to be non-limiting.

In addition, there are numerous other examples of contemplated, targetbiomolecules, such as, e.g., ADAM metalloproteinases (e.g. ADAM-9,ADAM-10, ADAM-12, ADAM-15, ADAM-17), ADP-ribosyltransferases (ART1,ART4), antigen F4/80, bone marrow stroma antigens (BST1, BST2), breakpoint cluster region-c-abl oncogene (BCR-ABL) proteins, C3aR (complementcomponent 3a receptors), CD7, CD13, CD14, CD15 (Lewis X orstage-specific embryonic antigen 1), CD23 (FC epsilon RII), CD45(protein tyrosine phosphatase receptor type C), CD49d, CD53, CD54(intercellular adhesion molecule 1), CD63 (tetraspanin), CD69, CD80,CD86, CD88 (complement component 5a receptor 1), CD115 (colonystimulating factor 1 receptor), IL-1R (interleukin-1 receptor), CD123(interleukin-3 receptor), CD129 (interleukin 9 receptor), CD183(chemokine receptor CXCR3), CD191 (CCR1), CD193 (CCR3), CD195 (chemokinereceptor CCR5), CD203c, CD225 (interferon-induced transmembrane protein1), CD244 (Natural Killer Cell Receptor 2B4), CD282 (Toll-like receptor2), CD284 (Toll-like receptor 4), CD294 (GPR44), CD305(leukocyte-associated immunoglobulin-like receptor 1), ephrin type-Areceptor 2 (EphA2), FceRIa, galectin-9, alpha-fetoprotein antigen 17-A1protein, human aspartyl (asparaginyl) beta-hydroxylase (HAAH),immunoglobulin-like transcript ILT-3, lysophosphatidlglycerolacyltransferase 1 (LPGAT1/IAA0205), lysosome-associated membraneproteins (LAMPs, such as CD107), melanocyte protein PMEL (gp100),myeloid-related protein-14 (mrp-14), NKG2D ligands (e.g., MICA, MICB,ULBP1, ULBP2, UL-16-binding proteins, H-60s, Rae-1s, and homologsthereof), receptor tyrosine-protein kinase erbB-3, SART proteins,scavenger receptors (such as CD64 and CD68), Siglecs (sialicacid-binding immunoglobulin-type lectins), syndecans (such as SDC1 orCD138), tyrosinase, tyrosinease-related protein 1 (TRP-1),tyrosinease-related protein 2 (TRP-2), tyrosinase associated antigen(TAA), APO-3, BCMA, CD2, CD3, CD4, CD8, CD18, CD27, CD28, CD29, CD41,CD49, CD90, CD95 (Fas), CD103, CD104, CD134 (OX40), CD137 (4-1BB), CD152(CTLA-4), chemokine receptors, complement proteins, cytokine receptors,histocompatibility proteins, ICOS, interferon-alpha, interferon-beta,c-myc, osteoprotegerin, PD-1, RANK, TACI, TNF receptor superfamilymember (TNF-R1, TNFR-2), Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4(see Scott A et al., Cancer Immunity 12: 14 (2012); Cheever M et al.,Clin Cancer Res 15: 5323-37 (2009)), for target biomolecules and notethe target biomolecules described therein are non-limiting examples).

In certain embodiments, the binding region comprises or consistsessentially of an immunoglobulin-type binding region capable ofspecifically binding with high-affinity to the cellular surface of acell type of the immune system. For example, immunoglobulin-type bindingdomains are known which bind to immune cell surface factors, such as,e.g., CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12,CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24,CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD33, CD34, CD35, CD36, CD37,CD38, CD40, CD41, CD56, CD61, CD62, CD66, CD95, CD 117, CD123, CD235,CD146, CD326, interleukin-1 receptor (IL-1R), interleukin-2 receptor(IL-2R), receptor activator of nuclear factor kappa B (RANKL),SLAM-associated protein (SAP), and TNFSF18 (tumor necrosis factor ligand18 or GITRL).

For further examples of target biomolecules and binding regionsenvisioned for use in the molecules of the present invention, see WO2005/092917, WO 2007/033497, US2009/0156417, JP4339511, EP1727827,DE602004027168, EP1945660, JP4934761, EP2228383, US2013/0196928, WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, US20150259428, 62/168,758,62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, andPCT/US2016/016580.

It will be appreciated by the skilled worker that any desired targetbiomolecule may be used to design or select a suitable binding region tobe associated and/or coupled with a Shiga toxin effector polypeptide toproduce a cell-targeting molecule of the present invention.

Any of the above binding regions described herein may be used alone orin combination with each individual embodiment of the present invention,including methods of the present invention.

The general structure of the cell-targeting molecules of the presentinvention is modular, in that various, diverse, cell-targeting bindingregions may be associated with various, Shiga toxin effectorpolypeptides of the present invention to create different,cell-targeting molecules of the present invention which exhibitdifferences in their cell-targeting activities due to differences intheir binding regions. This enables a variety of cell-targetingactivities to be exhibited by different embodiments of thecell-targeting molecules of the present invention such that differentembodiments target different types of cells with Shiga toxin effectorfunctions, such as, e.g., cytostasis, cytotoxicity, and intracellulardelivery of exogenous materials.

Furthermore, certain embodiments of the cell-targeting molecules of thepresent invention exhibit certain characteristics due to differences intheir respective Shiga toxin effector polypeptide regions, such as,e.g., low antigenicity and/or immunogenicity when administered to achordate, resistance to proteolytic cleavage by certain proteases, highstability when administered to a multicellular organism, in vivotolerability at high dosages, ability to deliver a cargo to anintracellular location, and/or ability to deliver a T-cell epitope to aMHC class I molecule for presentation on a cellular surface.

For the purposes of the present invention, the specific order ororientation of the Shiga toxin effector polypeptide region and thecell-targeting, binding region is not fixed in relation to each other orwithin the cell-targeting molecule of the present invention unlessexpressly noted. For example, when the cell-targeting molecule of thepresent invention is a fusion protein with an amino-terminal(s) andcarboxy-terminal(s), various arrangements of the components of theinvention may be suitable (see e.g. FIG. 1). In certain embodiments ofthe cell-targeting molecules of the present invention, the arrangementof their components in relation to each other or within thecell-targeting molecule are limited as described herein. For example,certain endoplasmic reticulum retention/retrieval signal motifs arecommonly positioned on a carboxy-terminus of a cell-targeting moleculeof the present invention and/or a carboxy-terminus of a proteincomponent of a cell-targeting molecule of the present invention.

C. Endoplasmic Reticulum Retention/Retrieval Signal Motif of a Member ofthe KDEL Family

Certain embodiments of the cell-targeting molecules of the presentinvention comprise one or more carboxy-terminal, endoplasmic reticulumretention/retrieval signal motifs of a member of the KDEL Family. Anyendoplasmic reticulum retention/retrieval signal motif described in WO2015/138435 may be suitable for use as a component of certaincell-targeting molecules of the present invention.

For purposes of the present invention, the phrase “endoplasmic reticulumretention/retrieval signal motif,” KDEL-type signal motif (“KDEL”disclosed as SEQ ID NO: 514), or signal motif refers to any member ofthe KDEL family capable of functioning within a eukaryotic cell topromote subcellular localization of a cell-targeting molecule of thepresent invention or component thereof to the endoplasmic reticulum viaKDEL receptors.

The carboxy-terminal lysine-asparagine-glutamate-leucine (KDEL) sequence(SEQ ID NO:514) is a canonical, endoplasmic reticulum retention andretrieval signal motif for soluble proteins in eukaryotic cells and isrecognized by the KDEL receptors (see, Capitani M, Sallese M, FEBS Lett583: 3863-71 (2009), for review). The KDEL family of signal motifsincludes many KDEL-like motifs, such as HDEL (SEQ ID NO:515), RDEL (SEQID NO:516), WDEL (SEQ ID NO:517), YDEL (SEQ ID NO:518), HEEL (SEQ IDNO:519), KEEL (SEQ ID NO:520), REEL (SEQ ID NO:521), KFEL (SEQ IDNO:522), KIEL (SEQ ID NO:523), DKEL (SEQ ID NO:524), KKEL (SEQ IDNO:525), HNEL (SEQ ID NO:526), HTEL (SEQ ID NO:527), KTEL (SEQ IDNO:528), and HVEL (SEQ ID NO:529), all of which are found at thecarboxy-terminals of proteins which are known to be residents of thelumen of the endoplasmic reticulum of throughout multiple phylogenetickingdoms (Munro S, Pelham H, Cell 48: 899-907 (1987); Raykhel I et al.,J Cell Biol 179: 1193-204 (2007)). The KDEL signal motif family includesat least 46 polypeptide variants shown using synthetic constructs(Raykhel, J Cell Biol 179: 1193-204 (2007)). Additional KDEL signalmotifs include ALEDEL (SEQ ID NO:530), HAEDEL (SEQ ID NO:531), HLEDEL(SEQ ID NO:532), KLEDEL (SEQ ID NO:533), IRSDEL (SEQ ID NO:534), ERSTEL(SEQ ID NO:535), and RPSTEL (SEQ ID NO:536) (Alanen H et al., J Mol Biol409: 291-7 (2011)). A generalized consensus motif representing themajority of KDEL signal motifs has been described as [KRHQSA]-[DENQ]-E-L(Hulo N et al., Nucleic Acids Res 34: D227-30 (2006)).

Proteins containing KDEL family signal motifs are bound by KDELreceptors distributed throughout the Golgi complex and transported tothe endoplasmic reticulum by a microtubule-dependent mechanism forrelease into the lumen of the endoplasmic reticulum (Griffiths G et al.,J Cell Biol 127: 1557-74 (1994); Miesenbock G, Rothman J, J Cell Biol129: 309-19 (1995)). KDEL receptors dynamically cycle between the Golgicomplex and endoplasmic reticulum (Jackson M et al., EMBO J. 9: 3153-62(1990); Schutze M et al., EMBO J. 13: 1696-1705 (1994)).

For purposes of the present invention, the members of the KDEL familyinclude synthetic signal motifs able to function within a eukaryoticcell to promote subcellular localization of a protein to the endoplasmicreticulum via KDEL receptors. In other words, some members of the KDELfamily might not occur in nature or have yet to be observed in naturebut have or may be constructed and empirically verified by the skilledworker using methods known in the art; see e.g., Raykhel I et al., JCell Biol 179: 1193-204 (2007).

As a component of certain cell-targeting molecules of the presentinvention, the KDEL-type signal motif is physically located, oriented,or arranged within the cell-targeting molecule such that it is on acarboxy-terminal of a polypeptide component of the cell-targetingmolecule of the present invention.

In certain embodiments of the cell-targeting molecules of the presentinvention, the binding region and the Shiga toxin effector polypeptideregion, and/or endoplasmic reticulum retention/retrieval signal motifmay be directly linked to each other and/or suitably linked to eachother via one or more intervening components, such as with one or morelinkers well known to the skilled worker and/or described herein.

D. Additional Exogenous Materials

In certain embodiments, the cell-targeting molecules of the presentinvention comprises an additional exogenous material. An “additionalexogenous material” as used herein refers to one or more atoms ormolecules, often not generally present in both Shiga toxins and nativetarget cells, where the cell-targeting molecule of the present inventioncan be used to specifically transport such material to the interior of acell. In one sense, the entire cell-targeting molecule of the inventionis an exogenous material which will enter the cell; thus, the“additional” exogenous materials are heterologous materials linked tobut other than the core cell-targeting molecule itself. Non-limitingexamples of additional exogenous materials are radionucleides, peptides,detection promoting agents, proteins, small molecule chemotherapeuticagents, and polynucleotides.

In certain embodiments of the cell-targeting molecules of the presentinvention, the additional exogenous material is one or moreradionucleides, such as, e.g., ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹¹¹In, ¹⁸⁶Re,¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P ⁶⁰C, and/or radioactive isotopes of lutetium.

In certain embodiments, the additional exogenous material comprises aproapoptotic peptide, polypeptide, or protein, such as, e.g., BCL-2,caspases (e.g. fragments of caspase-3 or caspase-6), cytochromes,granzyme B, apoptosis-inducing factor (AIF), BAX, tBid (truncated Bid),and proapoptotic fragments or derivatives thereof (see e.g., Ellerby Het al., Nat Med 5: 1032-8 (1999); Mai J et al., Cancer Res 61: 7709-12(2001); Jia L et al, Cancer Res 63: 3257-62 (2003); Liu Y et al., MolCancer Ther 2: 1341-50 (2003); Perea S et al., Cancer Res 64: 7127-9(2004); Xu Y et al., J Immunol 173: 61-7 (2004); Dalken B et al., CellDeath Differ 13: 576-85 (2006); Wang T et al., Cancer Res 67: 11830-9(2007); Kwon M et al., Mol Cancer Ther 7:1514-22 (2008); Qiu X et al.,Mol Cancer Ther 7: 1890-9 (2008); Shan L et al., Cancer Biol Ther 11:1717-22 (2008); Wang F et al., Clin Cancer Res 16: 2284-94 (2010); Kim Jet al., J Virol 85: 1507-16 (2011)).

In certain embodiments, the additional exogenous material comprises aprotein or polypeptide comprising an enzyme. In certain otherembodiments, the additional exogenous material is a nucleic acid, suchas, e.g. a ribonucleic acid that functions as a small inhibiting RNA(siRNA) or microRNA (miRNA). In certain embodiments, the additionalexogenous material is an antigen, such as antigens derived frompathogens, bacterial proteins, viral proteins, proteins mutated incancer, proteins aberrantly expressed in cancer, or T-cell complementarydetermining regions. For example, exogenous materials include antigens,such as those characteristic of antigen-presenting cells infected bybacteria, and T-cell complementary determining regions capable offunctioning as exogenous antigens. Exogenous materials comprisingpolypeptides or proteins may optionally comprise one or more antigenswhether known or unknown to the skilled worker.

In certain embodiments of the cell-targeting molecules of the presentinvention, all heterologous antigens and/or epitopes associated with theShiga toxin effector polypeptide are arranged in the cell-targetingmolecule amino-terminal to the carboxy-terminus of the Shiga toxin A1fragment region of the Shiga toxin effector polypeptide. In certainfurther embodiments, all heterologous antigens and/or epitopesassociated with the Shiga toxin effector polypeptide are associated,either directly or indirectly, with the Shiga toxin effector polypeptideat a position amino-terminal to the carboxy-terminus of the Shiga toxinA1 fragment region of the Shiga toxin effector polypeptide. In certainfurther embodiments, all additional exogenous material(s) which is anantigen is arranged amino-terminal to the Shiga toxin effectorpolypeptide, such as, e.g., fused directly or indirectly to the aminoterminus of the Shiga toxin effector polypeptide.

In certain embodiments of the cell-targeting molecules of the presentinvention, the additional exogenous material is a cytotoxic agent, suchas, e.g., a small molecule chemotherapeutic agent, anti-neoplasticagent, cytotoxic antibiotic, alkylating agent, antimetabolite,topoisomerase inhibitor, and/or tubulin inhibitor. Non-limiting examplesof cytotoxic agents suitable for use with the present invention includeaziridines, cisplatins, tetrazines, procarbazine, hexamethylmelamine,vinca alkaloids, taxanes, camptothecins, etoposide, doxorubicin,mitoxantrone, teniposide, novobiocin, aclarubicin, anthracyclines,actinomycin, amanitin, amatoxins, bleomycin, centanamycin(indolecarboxamide), plicamycin, mitomycin, daunorubicin, epirubicin,idarubicins, dolastatins, maytansines, maytansionoids, duromycin,docetaxel, duocarmycins, adriamycin, calicheamicin, auristatins,pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers (PBDs),carboplatin, 5-fluorouracil (5-FU), capecitabine, mitomycin C,paclitaxel, 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), rifampicin,cisplatin, methotrexate, gemcitabine, aceglatone, acetogenins (e.g.bullatacin and bullatacinone), aclacinomysins, AG1478, AG1571,aldophosphamide glycoside, alkyl sulfonates (e.g., busulfan,improsulfan, and piposulfan), alkylating agents (e.g. thiotepa andcyclosphosphamide), aminolevulinic acid, aminopterin, amsacrine,ancitabine, anthramycin, arabinoside, azacitidine, azaserine, aziridines(e.g., benzodopa, carboquone, meturedopa, and uredopa), azauridine,bestrabucil, bisantrene, bisphosphonates (e.g. clodronate), bleomycins,bortezomib, bryostatin, cactinomycin, callystatin, carabicin,carminomycin, carmofur, carmustine, carzinophilin, CC-1065,chlorambucil, chloranbucil, chlornaphazine, chlorozotocin,chromomycinis, chromoprotein enediyne antibiotic chromophores, CPT-11,cryptophycins (e.g. cryptophycin 1 and cryptophycin 8),cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunomycin,defofamine, demecolcine, detorubicin, diaziquone,6-diazo-5-oxo-L-norleucine, dideoxyuridine, difluoromethylornithine(DMFO), doxifluridine, doxorubicins (e.g., morpholinodoxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin, anddeoxydoxorubicin), dynemicins, edatraxate, edatrexate, eleutherobins,elformithine, elliptinium acetate, enediyne antibiotics (e.g.calicheamicins), eniluracil, enocitabine, epirubicins, epothilone,esorubicins, esperamicins, estramustine, ethylenimines,2-ethylhydrazide, etoglucid, fludarabine, folic acid analogues (e.g.,denopterin, methotrexate, pteropterin, and trimetrexate), folic acidreplenishers (e.g. frolinic acid), fotemustine, fulvestrant, gacytosine,gallium nitrate, gefitinib, gemcitabine, hydroxyurea, ibandronate,ifosfamide, imatinib mesylate, erlotinib, fulvestrant, letrozole,PTK787/ZK 222584 (Novartis, Basel, CH), oxaliplatin, leucovorin,rapamycin, lapatinib, lonafarnib, sorafenib, methylamelamines (e.g.,altretamine, triethy lenemelamine, triethy lenephosphoramide,triethylenethiophosphoramide and trimethylomelamine), pancratistatins,sarcodictyins, spongistatins, nitrogen mustards (e.g., chlorambucil,chlornaphazine, cyclophosphamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, and uracil mustard), nitrosureas (e.g., carmustine,fotemustine, lomustine, nimustine, and ranimnustine), dynemicins,neocarzinostatin chromophores, anthramycin, detorubicin, epirubicins,marcellomycins, mitomycins (e.g. mitomycin C), mycophenolic acid,nogalamycins, olivomycins, peplomycins, potfiromycins, puromycins,quelamycins, rodorubicins, ubenimex, zinostatins, zorubicins, purineanalogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, andthioguanine), pyrimidine analogs (e.g., ancitabine, azacitidine,6-azauridine, dideoxyuridine, doxifluridine, enocitabine, andfloxuridine), aceglatone, lentinan, lonidainine, maytansinoids (e.g.maytansins and ansamitocins), mitoguazone, mitoxantrone, mopidanmol,nitraerine, pentostatin, phenamet, pirarubicin, podophyllinic acid,2-ethylhydrazide, rhizoxin, sizofuran, spirogermanium, tenuazonic acid,triaziquone, 2,2′, 2″trichlorotriethylamine, trichothecenes (e.g., T-2toxin, verracurin A, roridin A, and anguidine), urethan, vindesine,mannomustine, mitobronitol, mitolactol, pipobroman, arabinoside,cyclophosphamide, toxoids (e.g. paclitaxel and doxetaxel),6-thioguanine, mercaptopurine, platinum, platinum analogs (e.g.cisplatin and carboplatin), etoposide (VP-16), mitoxantrone,vinorelbine, novantrone, daunomycin, xeloda, topoisomerase inhibitor RFS2000, retinoids (e.g. retinoic acid), capecitabine, lomustine,losoxantrone, mercaptopurines, nimustine, nitraerine, rapamycin,razoxane, roridin A, spongistatins, streptonigrins, streptozocins,sutent, T-2 toxin, thiamiprine, thiotepa, toxoids (e.g. paclitaxel anddoxetaxel), tubercidins, verracurin A, vinblastine, vincristine, andstructural analogs of any of the aforementioned (e.g. syntheticanalogs), and/or derivatives of any of the aforementioned (see e.g.,Lindell T et al., Science 170: 447-9 (1970); Remillard S et al., Science189: 1002-5 (1975); Ravry M et al., Am J Clin Oncol 8: 148-50 (1985);Ravry M et al., Cancer Treat Rep 69: 1457-8 (1985); Sternberg C et al.,Cancer 64: 2448-58 (1989); Bai R et al., Biochem Pharmacol 39: 1941-9(1990); Boger D, Johnson D, Proc Natl Acad Sci USA 92: 3642-9 (1995);Beck J et al., Leuk Lymphoma 41: 117-24 (2001); Cassady J et al., ChemPharm Bull (Tokyo) 52: 1-26 (2004); Sapra P et al., Clin Cancer Res 11:5257-64 (2005); Okeley N et al., Clinc Cancer Res 16: 888-97 (2010);Oroudjev E et al., Mol Cancer Ther 9: 2700-13 (2010); Ellestad G,Chirality 23: 660-71 (2011); Kantarjian H et al., Lancet Oncol 13:403-11 (2012); Moldenhauer G et al., J Natl Cancer Inst 104: 622-34(2012); Meulendijks D et al., Invest New Drugs 34: 119-28 (2016)).

E. Structure-Function Relationships of Cell-Targeting Molecules of theInvention

For certain embodiments of the cell-targeting molecules of the presentinvention, there specific structure-function relationships that havebeen observed, such as, e.g., component relative orientation effects oncytotoxic potency; furin-cleavage sensitivity effects on in vivotolerability at certain dosages; furin-cleavage sensitivity effects onin vitro stability; furin-cleavage sensitivity effects on in vivohalf-life; and furin-cleavage sensitivity effects on in vivo,non-specific toxicity in multicellular organisms.

In certain embodiments of the cell-targeting molecules of the presentinvention, the specific order or orientation of the Shiga toxin effectorpolypeptide region and binding region is fixed such that the bindingregion is located within the cell-targeting molecules more proximal tothe carboxy-terminus of the Shiga toxin effector polypeptide region thanto the amino-terminus of the Shiga toxin effector polypeptide region. Incertain embodiments of the cell-targeting molecules of the presentinvention, the arrangement of the Shiga toxin effector polypeptideregion within the cell-targeting molecule is limited to being at and/orproximal to the amino-terminus of a polypeptide component of thecell-targeting molecule (see FIG. 1). For example, certain embodimentsof the cell-targeting molecule of the present invention comprise 1) abinding region oriented within the cell-targeting molecule at a positioncarboxy-terminal to the Shiga toxin effector polypeptide region, 2) abinding region associated with the Shiga toxin effector polypeptideregion at a position distal from the amino-terminus of the Shiga toxineffector polypeptide region (e.g. distances of 50, 100, 200, or 250amino acid residues or greater), 3) a binding region not stericallycovering the amino-terminus of the Shiga toxin effector polypeptideregion, and/or 4) a binding region not sterically hindering astructure(s) near the amino-terminus of the Shiga toxin effectorpolypeptide region (see e.g. FIG. 1; WO 2015138452). In certain furtherembodiments, the cell-targeting molecules of the present invention arecapable of exhibiting more optimal cytotoxic potency, such as, e.g.,exhibiting a CD₅₀ value which is 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, or higher than a related cell-targetingreference molecule comprising the same Shiga toxin A Subunit effectorpolypeptide region(s) and binding region(s), wherein the binding regionis 1) amino-terminal to the Shiga toxin A Subunit effector polypeptideregion, 2) associated with the Shiga toxin effector polypeptide regionat a position proximal to the amino-terminus of the Shiga toxin effectorpolypeptide region (e.g. distances of less than 50, 40, 30, 20, or 10amino acid residues or less), 3) not sterically covering theamino-terminus of the Shiga toxin effector polypeptide region, and/or 4)not sterically hindering a structure(s) near the amino-terminus of theShiga toxin effector polypeptide region (see e.g. FIG. 1; WO2015/138452).

In certain embodiments, the Shiga toxin A Subunit effector polypeptideof the present invention comprises a Shiga toxin A1 fragment derivedregion comprising a disrupted furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment derived region (such asa disrupted furin-cleavage site located at the carboxy-terminus of aShiga toxin A1 fragment region) (see e.g. FIG. 1; WO 2015/191764). Incertain further embodiments, the Shiga toxin effector polypeptide ismore furin-cleavage resistant as compared to a related referencemolecule, such as, e.g., a molecule comprising a wild-type, Shiga toxinA Subunit or Shiga toxin A1 fragment (see e.g. WO 2015/191764). Incertain further embodiments, the Shiga toxin effector polypeptide of thepresent invention exhibits a reduction in furin-cleavage reproduciblyobserved to be 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, orless (including 100% for no cleavage) than the furin-cleavage of areference molecule observed in the same assay under the same conditions.In certain further embodiments, the Shiga toxin effector polypeptide ismore cleavage resistant to a protease other than furin as compared to arelated reference molecule, such as, e.g., a molecule comprising awild-type, Shiga toxin A Subunit or Shiga toxin A1 fragment.

Certain cell-targeting molecules of the present invention exhibitcytotoxic potencies within 100-fold, 20-fold, 10-fold, 5-fold, or lessthan a reference molecule comprising a wild-type Shiga toxin effectorpolypeptide region despite the lack of any compensatory structuralfeature for the disrupted furin-cleavage motif in the Shiga toxineffector polypeptide. For cell-targeting molecules comprising Shigatoxin A Subunit derived regions which do not maintain the furin cleavageevent, i.e. molecules comprising Shiga toxin A Subunit derivedcomponents which are not cleaved by furin inside target cells, onealternative for preserving maximal cytotoxicity is compensation.Compensation for the lack of furin cleavage of a Shiga toxin A Subunitregion in cytotoxic molecule might be accomplished by presenting theShiga toxin A Subunit region in a “pre-processed” form. For example, acell-targeting molecule comprising a Shiga toxin A Subunit region may beconstructed such that the carboxy-terminus of the Shiga toxin A Subunitderived polypeptide is 1) proximal to a carboxy-terminus of the moleculeand 2) matches or resembles a native Shiga toxin A1 fragment aftercleavage by furin (see WO 2015/191764). Such compensation is notrequired in certain cell-targeting molecules of the present invention,rather it is intentionally avoided in order to provide one or morefunction(s), such as, e.g., improved in vivo tolerability at certaindosages; increased in vitro stability; increased in vivo half-life;and/or reduced in vivo, non-specific toxicity in multicellularorganisms. For certain embodiments, these beneficial function(s) arepresent without any significant reduction in cytotoxic potency of thecell-targeting molecule of the present invention as compared to areference molecule comprising a wild-type Shiga toxin effectorpolypeptide.

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a Shiga toxin A Subunit effector polypeptidecomprising a Shiga toxin A1 fragment derived region comprising adisrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived region (such as a disrupted furin-cleavagesite located at the carboxy-terminus of a Shiga toxin A1 fragmentregion) (see e.g. FIG. 1; WO 2015/191764) but do not comprise anycompensatory protease cleavage site proximal to the carboxy-terminus ofthe Shiga toxin A1 fragment derived region and/or oriented between theShiga toxin effector polypeptide and a relatively large, molecule moiety(e.g. a binding region of a size greater than 4.5 kDa, 6, kDa, 9 kDa, 12kDa, 15 kDa, 20 kDa, 25 kDa, 28 kDa, 30 kDa, 41 kDa, or 50 kDa). Incertain further embodiments, the cell-targeting molecule of the presentinvention comprises a Shiga toxin effector polypeptide which is morefurin-cleavage resistant as compared to a related reference molecule,such as, e.g., a molecule comprising a wild-type, Shiga toxin A Subunitor Shiga toxin A1 fragment (see e.g. WO 2015/191764). In certain furtherembodiments, the cell-targeting molecule of the present inventionexhibits a reduction in furin-cleavage of 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 97%, 98%, 99%, or 100% less than the furin-cleavage of areference molecule observed in the same assay under the same conditionswhile the cell-targeting molecule exhibits a cytotoxic potency within100-fold, 20-fold, 10-fold, 5-fold, or less than a reference moleculecomprising a wild-type Shiga toxin effector polypeptide region. Incertain further embodiments, the cell-targeting molecule of the presentinvention exhibits an improvement in in vivo tolerability as compared toa related reference molecule comprising a Shiga toxin effectorpolypeptide having a wild-type furin cleavage motif and/or wild-typefurin cleavage site at the carboxy-terminus of its Shiga toxin A1fragment region (see e.g. WO 2015/191764). For example, an increase inin vivo tolerability may be determined by comparing measurements ofmortality, signs of morbidity, and/or certain clinical signs in groupsof laboratory animals administered different molecules at the samedosages (see e.g. Examples, infra; WO 2015/191764).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a Shiga toxin A Subunit effector polypeptidecomprising a Shiga toxin A1 fragment derived region comprising adisrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived region (such as a disrupted furin-cleavagesite located at the carboxy-terminus of a Shiga toxin A1 fragmentderived region) (see e.g. FIG. 1; WO 2015/191764). For certain furtherembodiments, the cell-targeting molecule of the present invention thatcomprise a cytotoxic component, the cell-targeting molecule exhibitsreduced non-specific toxicity as compared to more protease-cleavagesensitive variants, which have greater propensity to break apart andthereby release the cytotoxic component from the binding region,especially when administered to living materials, such as, e.g., apopulation of cells, a tissue, and/or an organism. Furthermore, certainprotease-cleavage resistant, cell-targeting molecules of the presentinvention may exhibit increased, in vivo, half-lives afteradministration to living materials (e.g., certain chordates) as comparedto more protease-cleavage sensitive variants based on theprotease-cleavage resistance conferred to the cell-targeting molecule bythe disrupted furin-cleavage motif at the carboxy-terminus of the Shigatoxin A1 fragment derived region.

III. Linkages Connecting Components of the Invention and/or theirSubcomponents

Individual cell-targeting binding regions, Shiga toxin effectorpolypeptides, and/or components of the cell-targeting molecules presentinvention may be suitably linked to each other via one or more linkerswell known in the art and/or described herein. Individual polypeptidesubcomponents of the binding regions, e.g. heavy chain variable regions(V_(H)), light chain variable regions (V_(L)), CDR, and/or ABR regions,may be suitably linked to each other via one or more linkers well knownin the art and/or described herein. Proteinaceous components of theinvention, e.g., multi-chain binding regions, may be suitably linked toeach other or other polypeptide components of the invention via one ormore linkers well known in the art. Peptide components of the invention,e.g., KDEL family endoplasmic reticulum retention/retrieval signalmotifs, may be suitably linked to another component of the invention viaone or more linkers, such as a proteinaceous linker, which are wellknown in the art.

Suitable linkers are generally those which allow each polypeptidecomponent of the present invention to fold with a three-dimensionalstructure very similar to the polypeptide components producedindividually without any linker or other component. Suitable linkersinclude single amino acids, peptides, polypeptides, and linkers lackingany of the aforementioned, such as various non-proteinaceous carbonchains, whether branched or cyclic.

Suitable linkers may be proteinaceous and comprise one or more aminoacids, peptides, and/or polypeptides. Proteinaceous linkers are suitablefor both recombinant fusion proteins and chemically linked conjugates. Aproteinaceous linker typically has from about 2 to about 50 amino acidresidues, such as, e.g., from about 5 to about 30 or from about 6 toabout 25 amino acid residues. The length of the linker selected willdepend upon a variety of factors, such as, e.g., the desired property orproperties for which the linker is being selected. In certainembodiments, the linker is proteinaceous and is linked near the terminusof a protein component of the present invention, typically within about20 amino acids of the terminus.

Suitable linkers may be non-proteinaceous, such as, e.g. chemicallinkers. Various non-proteinaceous linkers known in the art may be usedto link cell-targeting binding regions to the Shiga toxin effectorpolypeptide components of the cell-targeting molecules of the presentinvention, such as linkers commonly used to conjugate immunoglobulinpolypeptides to heterologous polypeptides. For example, polypeptideregions may be linked using the functional side chains of their aminoacid residues and carbohydrate moieties such as, e.g., a carboxy, amine,sulfhydryl, carboxylic acid, carbonyl, hydroxyl, and/or cyclic ringgroup. For example, disulfide bonds and thioether bonds may be used tolink two or more polypeptides. In addition, non-natural amino acidresidues may be used with other functional side chains, such as ketonegroups. Examples of non-proteinaceous chemical linkers include but arenot limited to N-succinimidyl (4-iodoacetyl)-aminobenzoate,S—(N-succinimidyl) thioacetate (SATA),N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio) toluene (SMPT),N-succinimidyl 4-(2-pyridyldithio)-pentanoate (SPP), succinimidyl4-(N-maleimidomethyl) cyclohexane carboxylate (SMCC or MCC),sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio) toluene,sulfosuccinimidyl-6-(α-methyl-α-(pyridyldithiol)-toluamido) hexanoate,N-succinimidyl-3-(-2-pyridyldithio)-proprionate (SPDP), succinimidyl6(3(-(-2-pyridyldithio)-proprionamido) hexanoate, sulfosuccinimidyl6(3(-(-2-pyridyldithio)-propionamido) hexanoate, maleimidocaproyl (MC),maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB),3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), alpha-alkylderivatives, sulfoNHS-ATMBA (sulfosuccinimidylN-[3-(acetylthio)-3-methylbutyryl-beta-alanine]), sulfodichlorophenol,2-iminothiolane, 3-(2-pyridyldithio)-propionyl hydrazide, Ellman'sreagent, dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine.

Suitable linkers, whether proteinaceous or non-proteinaceous, mayinclude, e.g., protease sensitive, environmental redox potentialsensitive, pH sensitive, acid cleavable, photocleavable, and/or heatsensitive linkers.

Proteinaceous linkers may be chosen for incorporation into recombinantfusion cell-targeting molecules of the present invention. Forrecombinant fusion cell-targeting proteins of the invention, linkerstypically comprise about 2 to 50 amino acid residues, preferably about 5to 30 amino acid residues. Commonly, proteinaceous linkers comprise amajority of amino acid residues with polar, uncharged, and/or chargedresidues, such as, e.g., threonine, proline, glutamine, glycine, andalanine. Non-limiting examples of proteinaceous linkers includealanine-serine-glycine-glycine-proline-glutamate (ASGGPE) (SEQ IDNO:538), valine-methionine (VM), alanine-methionine (AM),AM(G_(2 to 4)S)_(x)AM (SEQ ID NO: 539) where G is glycine, S is serine,and x is an integer from 1 to 10.

Proteinaceous linkers may be selected based upon the properties desired.Proteinaceous linkers may be chosen by the skilled worker with specificfeatures in mind, such as to optimize one or more of the fusionmolecule's folding, stability, expression, solubility, pharmacokineticproperties, pharmacodynamic properties, and/or the activity of the fuseddomains in the context of a fusion construct as compared to the activityof the same domain by itself. For example, proteinaceous linkers may beselected based on flexibility, rigidity, and/or cleavability. Theskilled worker may use databases and linker design software tools whenchoosing linkers. In certain linkers may be chosen to optimizeexpression. In certain linkers may be chosen to promote intermolecularinteractions between identical polypeptides or proteins to formhomomultimers or different polypeptides or proteins to formheteromultimers. For example, proteinaceous linkers may be selectedwhich allow for desired non-covalent interactions between polypeptidecomponents of the cell-targeting molecules of the invention, such as,e.g., interactions related to the formation dimers and other higherorder multimers.

Flexible proteinaceous linkers are often greater than 12 amino acidresidues long and rich in small, non-polar amino acid residues, polaramino acid residues, and/or hydrophilic amino acid residues, such as,e.g., glycines, serines, and threonines. Flexible proteinaceous linkersmay be chosen to increase the spatial separation between componentsand/or to allow for intramolecular interactions between components. Forexample, various “GS” linkers are known to the skilled worker and arecomposed of multiple glycines and/or one or more serines, sometimes inrepeating units, such as, e.g., (G_(x)S)_(n)(SEQ ID NO:540),(S_(x)G)_(n)(SEQ ID NO:541), (GGGGS)_(n) (SEQ ID NO:542), and(G)_(n)(SEQ ID NO:543), in which x is 1 to 6 and n is 1 to 30.Non-limiting examples of flexible proteinaceous linkers includeGKSSGSGSESKS (SEQ ID NO:544), EGKSSGSGSESKEF (SEQ ID NO:545),GSTSGSGKSSEGKG (SEQ ID NO:546), GSTSGSGKSSEGSGSTKG (SEQ ID NO:547),GSTSGSGKPGSGEGSTKG (SEQ ID NO:548), SRSSG (SEQ ID NO:549), and SGSSC(SEQ ID NO:550).

Rigid proteinaceous linkers are often stiff alpha-helical structures andrich in proline residues and/or one or more strategically placedprolines. Rigid linkers may be chosen to prevent intramolecularinteractions between linked components.

Suitable linkers may be chosen to allow for in vivo separation ofcomponents, such as, e.g., due to cleavage and/or environment-specificinstability. In vivo cleavable proteinaceous linkers are capable ofunlinking by proteolytic processing and/or reducing environments oftenat a specific site within an organism or inside a certain cell type. Invivo cleavable proteinaceous linkers often comprise protease sensitivemotifs and/or disulfide bonds formed by one or more cysteine pairs. Invivo cleavable proteinaceous linkers may be designed to be sensitive toproteases that exist only at certain locations in an organism,compartments within a cell, and/or become active only under certainphysiological or pathological conditions (such as, e.g., involvingproteases with abnormally high levels, proteases overexpressed atcertain disease sites, and proteases specifically expressed by apathogenic microorganism). For example, there are proteinaceous linkersknown in the art which are cleaved by proteases present onlyintracellularly, proteases present only within specific cell types, andproteases present only under pathological conditions like cancer orinflammation, such as, e.g., R-x-x-R motif andAMGRSGGGCAGNRVGSSLSCGGLNLQAM (SEQ ID NO:551).

In certain embodiments of the cell-targeting molecules of the presentinvention, a linker may be used which comprises one or more proteasesensitive sites to provide for cleavage by a protease present within atarget cell. In certain embodiments of the cell-targeting molecules ofthe invention, a linker may be used which is not cleavable to reduceunwanted toxicity after administration to a vertebrate organism.

Suitable linkers may include, e.g., protease sensitive, environmentalredox potential sensitive, pH sensitive, acid cleavable, photocleavable,and/or heat sensitive linkers, whether proteinaceous ornon-proteinaceous (see e.g., Doronina S et al., Bioconjug Chem 17:114-24 (2003); Saito G et al., Adv Drug Deliv Rev 55: 199-215 (2003);Jeffrey S et al., J Med Chem 48: 1344-58 (2005); Sanderson R et al.,Clin Cancer Res 11: 843-52 (2005); Erickson H et al., Cancer Res 66:4426-33 (2006); Chen X et al., Adv Drug Deliv Rev 65: 1357-69 (2013)).Suitable cleavable linkers may include linkers comprising cleavablegroups which are known in the art.

Suitable linkers may include pH sensitive linkers. For example, certainsuitable linkers may be chosen for their instability in lower pHenvironments to provide for dissociation inside a subcellularcompartment of a target cell (see e.g., van Der Velden V et al., Blood97: 3197-204 (2001); Ulbrich K, Subr V, Adv Drug Deliv Rev 56: 1023-50(2004)). For example, linkers that comprise one or more trityl groups,derivatized trityl groups, bismaleimideothoxy propane groups, adipicacid dihydrazide groups, and/or acid labile transferrin groups, mayprovide for release of components of the cell-targeting molecules of theinvention, e.g. a polypeptide component, in environments with specificpH ranges. In certain linkers may be chosen which are cleaved in pHranges corresponding to physiological pH differences between tissues,such as, e.g., the pH of tumor tissue is lower than in healthy tissues.

Photocleavable linkers are linkers that are cleaved upon exposure toelectromagnetic radiation of certain wavelength ranges, such as light inthe visible range. Photocleavable linkers may be used to release acomponent of a cell-targeting molecule of the invention, e.g. apolypeptide component, upon exposure to light of certain wavelengths.Non-limiting examples of photocleavable linkers include a nitrobenzylgroup as a photocleavable protective group for cysteine,nitrobenzyloxycarbonyl chloride cross-linkers,hydroxypropylmethacrylamide copolymer, glycine copolymer, fluoresceincopolymer, and methylrhodamine copolymer. Photocleavable linkers mayhave particular uses in linking components to form cell-targetingmolecules of the invention designed for treating diseases, disorders,and conditions that can be exposed to light using fiber optics.

In certain embodiments of the cell-targeting molecules of the presentinvention, a cell-targeting binding region is linked to a Shiga toxineffector polypeptide of the present invention using any number of meansknown to the skilled worker, including both covalent and noncovalentlinkages.

In certain embodiments of the cell-targeting molecules of the presentinvention, the molecule comprises a binding region which is a scFv witha linker connecting a heavy chain variable (V_(H)) domain and a lightchain variable (V_(L)) domain. There are numerous linkers known in theart suitable for this purpose, such as, e.g., the 15-residue (Gly4Ser)₃peptide (SEQ ID NO:552). Suitable scFv linkers which may be used informing non-covalent multivalent structures include GGS (SEQ ID NO:553),GGGS (SEQ ID NO:554), GGGGS (SEQ ID NO:555), GGGGSGGG (SEQ ID NO:556),GGSGGGG (SEQ ID NO:557), GSTSGGGSGGGSGGGGSS (SEQ ID NO:558), andGSTSGSGKPGSSEGSTKG (SEQ ID NO:559).

Suitable methods for linkage of the components of the cell-targetingmolecules of the present invention may be by any method presently knownin the art for accomplishing such, so long as the attachment does notsubstantially impede the binding capability of the cell-targetingbinding region, the cellular internalization of the Shiga toxin effectorpolypeptide component, and/or when appropriate the desired Shiga toxineffector function(s) as measured by an appropriate assay, includingassays described herein.

For the purposes of the cell-targeting molecules of the presentinvention, the specific order or orientation is not fixed for thecomponents: the Shiga toxin effector polypeptide(s), the bindingregion(s), and any optional linker(s), in relation to each other or theentire cell-targeting molecule (see e.g. FIG. 1) unless specificallynoted. The components of the cell-targeting molecules of the presentinvention may be arranged in any order provided that the desiredactivity(ies) of the binding region and Shiga toxin effector polypeptideare not eliminated.

IV. Examples of Structural Variations of the Shiga Toxin EffectorPolypeptides and Cell-Targeting Molecules of the Invention

In certain embodiments, a Shiga toxin effector polypeptide of thepresent invention may comprise or consist essentially of a truncatedShiga toxin A Subunit.

Truncations of Shiga toxin A Subunits might result in the deletion of anentire epitope(s) and/or epitope region(s), B-cell epitopes, CD4+ T-cellepitopes, and/or furin-cleavage sites without affecting Shiga toxineffector functions, such as, e.g., catalytic activity and cytotoxicity.The smallest Shiga toxin A Subunit fragment shown to exhibit fullenzymatic activity was a polypeptide composed of residues 1-239 of Slt1A(LaPointe P et al., J Biol Chem 280: 23310-18 (2005)). The smallestShiga toxin A Subunit fragment shown to exhibit significant enzymaticactivity was a polypeptide composed of residues 75-247 of StxA (A1-JaufyA et al., Infect Immun 62: 956-60 (1994)).

Although Shiga toxin effector polypeptides of the present invention maycommonly be smaller than the full-length Shiga toxin A Subunit, it ispreferred that the Shiga toxin effector polypeptide region of acell-targeting molecule of the present invention maintain thepolypeptide region from amino acid position 77 to 239 (SLT-1A (SEQ IDNO:1) or StxA (SEQ ID NO:2)) or the equivalent in other A Subunits ofmembers of the Shiga toxin family (e.g. 77 to 238 of (SEQ ID NO:3)). Forexample, in certain embodiments of the molecules of the presentinvention, the Shiga toxin effector polypeptides of the presentinvention derived from SLT-1A may comprise or consist essentially ofamino acids 75 to 251 of SEQ ID NO:1, 1 to 241 of SEQ ID NO:1, 1 to 251of SEQ ID NO:1, or amino acids 1 to 261 of SEQ ID NO:1, wherein relativeto a wild-type Shiga toxin A Subunit at least one amino acid residue ismutated or has been deleted in an endogenous epitope and/or epitoperegion, and/or wherein there is a disrupted, furin-cleavage motif regionat the carboxy-terminus of a Shiga toxin A1 fragment derived region.Similarly, Shiga toxin effector polypeptide regions derived from StxAmay comprise or consist essentially of amino acids 75 to 251 of SEQ IDNO:2, 1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or amino acids 1to 261 of SEQ ID NO:2, wherein relative to a wild-type Shiga toxin ASubunit at least one amino acid residue is mutated or has been deletedin an endogenous epitope and/or epitope region, and/or wherein there isa disrupted, furin-cleavage motif region at the carboxy-terminus of aShiga toxin A1 fragment derived region. Additionally, Shiga toxineffector polypeptide regions derived from SLT-2 may comprise or consistessentially of amino acids 75 to 251 of SEQ ID NO:3, 1 to 241 of SEQ IDNO:3, 1 to 251 of SEQ ID NO:3, or amino acids 1 to 261 of SEQ ID NO:3,wherein relative to a wild-type Shiga toxin A Subunit at least one aminoacid residue is mutated or has been deleted in an endogenous epitopeand/or epitope region, and/or wherein there is a disrupted,furin-cleavage motif region at the carboxy-terminus of a Shiga toxin A1fragment derived region.

The invention further provides variants of Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention,wherein the Shiga toxin effector polypeptide differs from a naturallyoccurring Shiga toxin A Subunit by only or up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by nomore than that which retains at least 85%, 90%, 95%, 99% or more aminoacid sequence identity). Thus, a molecule of the present inventionderived from an A Subunit of a member of the Shiga toxin family maycomprise additions, deletions, truncations, or other alterations fromthe original sequence as long as at least 85%, 90%, 95%, 99% or moreamino acid sequence identity is maintained to a naturally occurringShiga toxin A Subunit and wherein relative to a wild-type Shiga toxin ASubunit at least one amino acid residue is mutated or has been deletedin an endogenous epitope and/or epitope region, and/or wherein there isa disrupted, furin-cleavage motif region at the carboxy-terminus of aShiga toxin A1 fragment derived region.

Accordingly, in certain embodiments, the Shiga toxin effectorpolypeptide of a molecule of the present invention comprises or consistsessentially of amino acid sequences having at least 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 99.7% overall sequenceidentity to a naturally occurring Shiga toxin A Subunit, such as SLT-1A(SEQ ID NO:1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) whereinrelative to a wild-type Shiga toxin A Subunit at least one amino acidresidue is mutated or has been deleted in an endogenous epitope and/orepitope region, and/or wherein there is a disrupted, furin-cleavagemotif region at the carboxy-terminus of a Shiga toxin A1 fragmentderived region.

Optionally, either a full-length or a truncated version of the Shigatoxin A Subunit may comprise the Shiga toxin effector polypeptide regionof a molecule of the present, wherein the Shiga toxin derivedpolypeptide comprises one or more mutations (e.g. substitutions,deletions, insertions, or inversions) as compared to a naturallyoccurring Shiga toxin. It is preferred in certain embodiments of theinvention that the Shiga toxin effector polypeptides have sufficientsequence identity to a naturally occurring Shiga toxin A Subunit toretain cytotoxicity after entry into a cell, either by well-knownmethods of host cell transformation, transfection, infection orinduction, or by internalization mediated by a cell-targeting bindingregion linked with the Shiga toxin effector polypeptide. The mostcritical residues for enzymatic activity and/or cytotoxicity in theShiga toxin A Subunits have been mapped to the followingresidue-positions: asparagine-75, tyrosine-77, glutamate-167,arginine-170, and arginine-176 among others (Di R et al., Toxicon 57:525-39 (2011)). In any one of the embodiments of the invention, theShiga toxin effector polypeptides may preferably but not necessarilymaintain one or more conserved amino acids at positions, such as thosefound at positions 77, 167, 170, and 176 in StxA, SLT-1A, or theequivalent conserved position in other members of the Shiga toxin familywhich are typically required for cytotoxic activity. The capacity of acytotoxic molecule of the invention to cause cell death, e.g. itscytotoxicity, may be measured using any one or more of a number ofassays well known in the art.

A. Examples of De-Immunized, Shiga Toxin Effector Polypeptides

In certain embodiments, the de-immunized, Shiga toxin effectorpolypeptide of the present invention may consist essentially of atruncated Shiga toxin A Subunit having two or more mutations.Truncations of Shiga toxin A Subunits might result in the deletion of anentire epitope(s) and/or epitope region(s), B-cell epitopes, CD4+ T-cellepitopes, and/or furin-cleavage sites without affecting Shiga toxineffector functions, such as, e.g., catalytic activity and cytotoxicity.Truncating the carboxy-terminus of SLT-1A, StxA, or SLT-2A to aminoacids 1-251 removes two predicted B-cell epitope regions, two predictedCD4 positive (CD4+) T-cell epitopes, and a predicted discontinuousB-cell epitope. Truncating the amino-terminus of SLT-1A, StxA, or SLT-2Ato 75-293 removes at least three predicted B-cell epitope regions andthree predicted CD4+ T-cell epitopes. Truncating both amino- andcarboxy-terminals of SLT-1A, StxA, or SLT-2A to 75-251 deletes at leastfive predicted B-cell epitope regions, four putative CD4+ T-cellepitopes and one predicted discontinuous B-cell epitope.

In certain embodiments, a de-immunized, Shiga toxin effector polypeptideof the present invention may comprise or consist essentially of afull-length or truncated Shiga toxin A Subunit with at least onemutation (relative to a wild-type Shiga toxin polypeptide), e.g.deletion, insertion, inversion, or substitution, in a provided,endogenous, B-cell and/or CD4+ T-cell epitope region. In certainembodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption which comprises a mutation (relative toa wild-type Shiga toxin polypeptide) which includes a deletion of atleast one amino acid residue within the endogenous, B-cell and/or CD4+T-cell epitope region. In certain embodiments, the Shiga toxin effectorpolypeptide of the present invention comprises a disruption whichcomprises an insertion of at least one amino acid residue within theendogenous, B-cell and/or CD4+ T-cell epitope region. In certainembodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption which comprises an inversion of aminoacid residues, wherein at least one inverted amino acid residue iswithin the endogenous, B-cell and/or CD4+ T-cell epitope region. Incertain embodiments, the Shiga toxin effector polypeptide of the presentinvention comprises a disruption which comprises a mutation (relative toa wild-type Shiga toxin polypeptide), such as, e.g., an amino acidsubstitution, an amino acid substitution to a non-standard amino acid,and/or an amino acid residue with a chemically modified side chain.Non-limiting examples of de-immunized, Shiga toxin effector sub-regionssuitable for use in the present invention are described in WO2015/113005, WO 2015/113007 and WO 2015/191764. Numerous, non-limitingexamples of Shiga toxin effector polypeptides of the present inventionwhich comprise amino acid substitutions are provided in the Examples.

In other embodiments, the de-immunized, Shiga toxin effector polypeptideof the present invention comprises a truncated Shiga toxin A Subunitwhich is shorter than a full-length Shiga toxin A Subunit wherein atleast one amino acid residue is disrupted in a natively positioned,B-cell and/or CD4+ T-cell epitope region provided in the Examples (seee.g. Tables 1-7 and/or Table B).

To create a de-immunized, Shiga toxin effector polypeptide, in principlemodifying any amino acid residue in a provided epitope region by variousmeans can result in a disruption of an epitope, such as, e.g., amodification which represents a deletion, insertion, inversion,rearrangement, substitution, and chemical modification of a side chainrelative to a wild-type Shiga toxin polypeptide. However, modifyingcertain amino acid residues and using certain amino acid modificationsare more likely to successfully reduce antigenicity and/orimmunogenicity while maintaining a certain level of a Shiga toxineffector function(s). For example, terminal truncations and internalamino acid substitutions are preferred because these types ofmodifications maintain the overall spacing of the amino acid residues ina Shiga toxin effector polypeptide and thus are more likely to maintainShiga toxin effector polypeptide structure and function.

Among certain embodiments of the present invention, the de-immunized,Shiga toxin effector polypeptide comprising or consisting essentially ofamino acids 75 to 251 of SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2),and/or SLT-2A (SEQ ID NO:3) wherein at least one amino acid residue isdisrupted in a natively positioned, epitope region provided in theExamples (see e.g. Tables 1-7 and/or 12). Among certain otherembodiments are de-immunized, Shiga toxin effector polypeptides whichcomprise or consist essentially of amino acids 1 to 241 of SLT-1A (SEQID NO:1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) wherein atleast one amino acid residue is disrupted in a natively positioned,epitope region provided in the Examples (see e.g. Tables 1-7 and/or 12).Further embodiments are de-immunized, Shiga toxin effector polypeptideswhich comprise or consist essentially of amino acids 1 to 251 of SLT-1A(SEQ ID NO:1), StxA (SEQ ID NO:2), and/or SLT-2A (SEQ ID NO:3) whereinat least one amino acid residue is disrupted in a natively positioned,epitope region provided in the Examples (see e.g. Tables 1-7 and/or 12).Further embodiments are Shiga toxin effector polypeptides comprisingamino acids 1 to 261 of SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:2), and/orSLT-2A (SEQ ID NO:3) wherein at least one amino acid residue isdisrupted in a natively positioned, epitope region provided in theExamples (see e.g. Tables 1-7 and/or 12).

There are numerous, diverse, internal amino acid substitutions that canbe used to create de-immunized, Shiga toxin effector polypeptides of theinvention. Of the possible substitute amino acids to use within anepitope region, the following substitute amino acid residues arepredicted to be the most likely to reduce the antigenicity and/orimmunogenicity of an epitope G, D, E, S, T, R, K, and H. Except forglycine, these amino acid residues may all be classified as polar and/orcharged residues. Of the possible amino acids to substitute with, thefollowing amino acids A, G, V, L, I, P, C, M, F, S, D, N, Q, H, and Kare predicted to be the most likely to reduce antigenicity and/orimmunogenicity while providing the retention of a significant level of aShiga toxin effector function(s), depending on the amino acidsubstituted for. Generally, the substitution should change a polarand/or charged amino acid residue to a non-polar and uncharged residue(see e.g. WO 2015/113005). In addition, it may be beneficial to epitopedisruption to reduce the overall size and/or length of the amino acidresidue's R-group functional side chain (see e.g. WO 2015/113005).However despite these generalities of substitutions most likely toconfer epitope disruption, because the aim is to preserve significantShiga toxin effector function(s), the substitute amino acid might bemore likely to preserve Shiga toxin effector function(s) if it resemblesthe amino acid substituted for, such as, e.g., a nonpolar and/oruncharged residue of similar size substituted for a polar and/or chargedresidue.

In the Examples below and in WO 2015/113005, many mutations have beenempirically tested for effect(s) on the Shiga toxin effector function ofvarious Shiga toxin effector polypeptides and cell-targeting molecules.Table B summarizes the results described in the Examples and in WO2015/113005 where an amino acid substitution, alone or in combinationwith one or more other substitutions, did not prevent the exhibition ofa potent level of a Shiga toxin effector function(s). Table B uses theepitope region numbering scheme described in the Examples below (seeExample 1-Table 7, infra).

TABLE B Amino Acid Substitutions in Shiga Toxin Effector PolypeptidesEpitope Region natively positioned amino acid positions DisruptedSubstitution B-Cell Epitope Region T-Cell Epitope 1 K1A  1-15 1 K1M 1-15 1 T4I  1-15  4-33 1 D6R  1-15  4-33 1 S8I  1-15  4-33 1 T9V  1-15 4-33 1 T9I  1-15  4-33 1 K11A  1-15  4-33 1 K11H  1-15  4-33 1 T12K 1-15  4-33 2 S33I 27-37  4-33 2 S33C 27-37  4-33 3 S43N 39-48 34-78 3G44L 39-48 34-78 3 T45V 39-48 34-78 3 T45I 39-48 34-78 3 S45V 39-4834-78 3 S45I 39-48 34-78 3 G46P 39-48 34-78 3 D47G 39-48 34-78 3 D47M39-48 34-78 3 N48V 39-48 34-78 3 N48F 39-48 34-78 — L49A immunogenicresidue 34-78 — F50T 34-78 — A51V 34-78 4 D53A 53-66 34-78 4 D53G 53-6634-78 4 D53N 53-66 34-78 4 V54L 53-66 34-78 4 V54I 53-66 34-78 4 R55A53-66 34-78 4 R55V 53-66 34-78 4 R55L 53-66 34-78 4 G56P 53-66 34-78 4I57M 53-66 34-78 4 I57F 53-66 34-78 4 D58A 53-66 34-78 4 D58V 53-6634-78 4 D58F 53-66 34-78 4 P59A 53-66 34-78 4 P59F 53-66 34-78 4 E60I53-66 34-78 4 E60T 53-66 34-78 4 E60R 53-66 34-78 4 E61A 53-66 34-78 4E61V 53-66 34-78 4 E61L 53-66 34-78 4 G62A 53-66 34-78 — R84A  77-103 —V88A  77-103 5 D94A  94-115  77-103 5 S96I  94-115  77-103 5 T104N 94-115 5 A105L  94-115 5 T107P  94-115 5 L108M  94-115 5 S109V  94-1155 G110A  94-115 5 D111T  94-115 5 S112V  94-115 6 D141A 141-153 128-1686 G147A 141-153 128-168 — V154A 128-168 7 R179A 179-190 160-183 7 T180G179-190 160-183 7 T181I 179-190 160-183 7 D183A 179-190 160-183 7 D183G179-190 160-183 7 D184A 179-190 7 D184F 179-190 7 L185V 179-190 7 S186A179-190 7 S186F 179-190 7 G187A 179-190 7 G187T 179-190 7 R188A 179-1907 R188L 179-190 7 S189A 179-190 — D198A immunogenic residue — R205Aimmunogenic residue — C242S 236-258 8 R248A 243-257 236-258 8 R251A243-257 236-258

Based on the empirical evidence in the Examples herein and in WO2015/113005, certain amino acid positions in the A Subunits of Shigatoxins are predicted to tolerate epitope disruptions while stillretaining significant Shiga toxin effector functions. For example, thefollowing natively occurring positions tolerate amino acidsubstitutions, either alone or in combination, while retaining a Shigatoxin effector function(s) such as cytotoxicity—1 of SEQ ID NO:1 or SEQID NO: 2; 4 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; 8 of SEQ IDNO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; 9 of SEQ ID NO: 1, SEQ ID NO: 2,or SEQ ID NO: 3; 11 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; 33of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 ofSEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQID NO:1 or SEQ ID NO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 ofSEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ IDNO:1 or SEQ ID NO:2; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ IDNO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ IDNO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1 or SEQ IDNO:2; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 96 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO: 3; 104 of SEQ ID NO:1 or SEQ ID NO:2;105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1 or SEQ ID NO:2;108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1 orSEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 orSEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2;186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 orSEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 orSEQ ID NO:2; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ ID NO: 3; 205of SEQ ID NO:1 or SEQ ID NO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 orSEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 orSEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2.

The empirical data in the Examples and in WO 2015/113005 point towardsother epitope disrupting substitutions and combinations of epitopedisrupting substitutions that can reduce antigenicity and/orimmunogenicity of a Shiga toxin effector polypeptide while retaining theability of the Shiga toxin effector polypeptide to exhibit a significantShiga toxin effector function such as, e.g., new combinations of theaforementioned truncations and positions tolerating substitutions aswell as new substitutions at identical positions or conserved positionsin related Shiga toxin A Subunits.

It is predictable that other amino acid substitutions to amino acidresidues of a conservative functional group of a substitution testedherein may reduce antigenicity and/or immunogenicity while preserving asignificant Shiga toxin effector function. For example, othersubstitutions known to the skilled worker to be similar to any of K1A,K1M, T4I, D6R, S81, T8V, T9I, S9I, K11A, K11H, T12K, S33I, S33C, S43N,G44L, S45V, S45I, T45V, T45I, G46P, D47M, D47G, N48V, N48F, L49A, F50T,A51V, D53A, D53N, D53G, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M,D58A, D58V, D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A,R84A, V88A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, T109V, G110A,D111T, S112V, D141A, G147A, V154A, R179A, T180G, T181I, D183A, D183G,D184A, D184A, D184F, L185V, L185D, S186A, S186F, G187A, G187T, R188A,R188L, S189A, D198A, R204A, R205A, C242S, S247I, Y247A, R248A, R250A,R251A, or D264A, G264A, T286A, and/or T2861 may disrupt an endogenousepitope while maintaining at least one Shiga toxin effector function. Inparticular, amino acid substitutions to conservative amino acid residuessimilar to K1A, K1M, T4I, S81, T8V, T9I, S9I, K11A, K11H, S33I, S33C,S43N, G44L, S45V, S45I, T45V, T45I, G46P, D47M, N48V, N48F, L49A, A51V,D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V,D58F, P59A, E601, E60T, E61A, E61V, E61L, G62A, R84A, V88A, D94A, S96I,T104N, T107P, L108M, S109V, T109V, G110A, D111T, S112V, D141A, G147A,V154A, R179A, T180G, T181I, D183A, D183G, D184A, D184F, L185V, S186A,S186F, G187A, R188A, R188L, S189A, D198A, R204A, R205A, C242S, S247I,Y247A, R248A, R250A, R251A, D264A, G264A, T286A, and T286I may have thesame or similar effects. In certain embodiments, a Shiga toxin effectorpolypeptide of the invention may comprise similar conservative aminoacid substitutions to empirically tested ones, such as, e.g., K1 to G,V, L, I, F, and H; T4 to A, G, V, L, F, M, and S; S8 to A, G, V, L, F,and M; T9 to A, G, L, F, M, and S; S9 to A, G, L, I, F, and M; K11 to G,V, L, I, F, and M; S33 to A, G, V, L, F, and M; S43 to A, G, V, L, I, F,and M; S45 to A, G, L, F, and M; T45 to A, G, L, F, and M; D47 to A, V,L, I, F, S, and Q; N48 to A, G, L, and M; L49 to G; Y49 to A; D53 to V,L, I, F, S, and Q; R55 to G, I, F, M, Q, S, K, and H; D58 to G, L, I, S,and Q; P59 to G; E60 to A, G, V, L, F, S, Q, N, D, and M; E61 to G, I,F, S, Q, N, D, M, and R; R84 to G, V, L, I, F, M, Q, S, K, and H; V88 toG; 188 to G; D94 to G, V, L, I, F, S, and Q; S96 to A, G, V, L, F, andM; T107 to A, G, V, L, I, F, M, and S; S107 to A, G, V, L, I, F, and M;S109 to A, G, I, L, F, and M; T109 to A, G, I, L, F, M, and S; Si 12 toA, G, L, I, F, and M; D141 to V, L, I, F, S, and Q; V154 to G; R179 toG, V, L, I, F, M, Q, S, K, and H; T180 to A, V, L, I, F, M, and S; T181to A, G, V, L, F, M, and S; D183 to V, L, I, F, S, and Q; D184 to G, V,L, I, S, and Q; S186 to G, V, I, L, and M; R188 to G, V, I, F, M, Q, S,K, and H; S189 to G, V, I, L, F, and M; D197 to V, L, I, F, S, and Q;D198 to A, V, L, I, F, S, and Q; R204 to G, V, L, I, F, M, Q, S, K, andH; R205 to G, V, L, I, F, M, Q, S, K and H; S247 to A, G, V, I, L, F,and M; Y247 to A, G, V, L, I, F, and M; R248 to G, V, L, I, F, M, Q, S,K, and H; R250 to G, V, L, I, F, M, Q, S, K, and H; R251 to G, V, L, I,F, M, Q, S, K, and H; D264 to A, G, V, L, I, F, S, and Q; and T286 to A,G, V, L, I, F, M, and S.

Similarly, amino acid substitutions which remove charge, polarity,and/or reduce side chain length can disrupt an epitope while maintainingat least one Shiga toxin effector function. In certain embodiments, aShiga toxin effector polypeptide of the invention may comprise one ormore epitopes disrupted by substitutions such that side chain charge isremoved, polarity is removed, and/or side chain length is reduced suchas, e.g., substituting the appropriate amino acid selected from thefollowing group A, G, V, L, I, P, C, M, F, S, D, N, Q, H, or K for theamino acid residue at position 1 of SEQ ID NO:1 or SEQ ID NO:2; 4 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO:1 or SEQ ID NO:2; 8of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 11 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3;12 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 33 of SEQ ID NO:1 orSEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1 or SEQ IDNO:2; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 49 of SEQ ID NO:1 or SEQ ID NO:2; 50 of SEQ ID NO:1 orSEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQID NO:2; 54 of SEQ ID NO:1 or SEQ ID NO:2; 55 of SEQ ID NO:1 or SEQ IDNO:2; 56 of SEQ ID NO:1 or SEQ ID NO:2; 57 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 58 of SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO:3; 59 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ IDNO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ IDNO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO:3; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 96 ofSEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO: 3; 104 of SEQ ID NO:1 or SEQ IDNO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2;111 of SEQ ID NO:1 or SEQ ID NO:2; 112 of SEQ ID NO:1, SEQ ID NO: 2, orSEQ ID NO:3; 141 of SEQ ID NO:1 or SEQ ID NO:2; 147 of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2;181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2;189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ IDNO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ IDNO:2; 241 of SEQ ID NO:3; 242 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQID NO:1 or SEQ ID NO: 2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 ofSEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ IDNO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, aShiga toxin effector polypeptide of the present invention may compriseone or more of the following amino acid substitutions: K1 to A, G, V, L,I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F,S, and Q; S8 to A, G, V, I, L, F, and M; T8 to A, G, V, I, L, F, M, andS; T9 to A, G, V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11to A, G, V, L, I, F, M and H; T12 to A, G, V, I, L, F, M, and S; S33 toA, G, V, L, I, F, and M; S43 to A, G, V, L, I, F, and M; G44 to A and L;S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46 to Aand P; D47 to A, G, V, L, I, F, S, and Q; N48 to A, G, V, L, and M; L49to A or G; F50; A51 to V; D53 to A, G, V, L, I, F, S, and Q; V54 to A,G, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P;157 to A, G, M, and F; L57 to A, G, M, and F; D58 to A, G, V, L, I, F,S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D, M,and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A; D94 toA, G, V, L, I, F, S, and Q; R84 to A, G, V, L, I, F, M, Q, S, K, and H;V88 to A and G; 188 to A, G, and V; D94; S96 to A, G, V, I, L, F, and M;T104 to A, G, V, I, L, F, M, and S; A105 to L; T107 to A, G, V, I, L, F,M, and S; S107 to A, G, V, L, I, F, and M; L108 to A, G, and M; S109 toA, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, and S; G110 to A;D111 to A, G, V, L, I, F, S, and Q; S112 to A, G, V, L, I, F, and M;D141 to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G; R179 toA, G, V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S;T181 to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q;D184 to A, G, V, L, I, F, S, and Q; L185 to A, G, and V; 5186 to A, G,V, I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, andH; S189 to A, G, V, I, L, F, and M; D197 to A, G, V, L, I, F, S, and Q;D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I, F, M, Q, S,K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; C242 to A, G, V,and S; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M;R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F,M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; C262 toA, G, V, and S; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286to A, G, V, L, I, F, M, and S.

In addition, any amino acid substitution in one epitope region of aShiga toxin effector polypeptide which disrupts an epitope whileretaining significant Shiga toxin effector function is combinable withany other amino acid substitution in the same or a different epitoperegion which disrupts an epitope while retaining significant Shiga toxineffector function to form a de-immunized, Shiga toxin effectorpolypeptide with multiple epitope regions disrupted while stillretaining a significant level of Shiga toxin effector function. Incertain embodiments, a Shiga toxin effector polypeptide of the inventionmay comprise a combinations of two or more of the aforementionedsubstitutions and/or the combinations of substitutions described in WO2015/113005.

Based on the empirical evidence in the Examples and in WO 2015/113005,certain amino acid regions in the A Subunits of Shiga toxins arepredicted to tolerate epitope disruptions while still retainingsignificant Shiga toxin effector functions. For example, the epitoperegions natively positioned at 1-15, 39-48, 53-66, 55-66, 94-115,180-190, 179-190, and 243-257 tolerated multiple amino acid substitutioncombinations simultaneously without compromising Shiga toxin enzymaticactivity and cytotoxicity.

B. Examples of Furin-Cleavage Resistant, Shiga Toxin EffectorPolypeptides

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention may comprise a disrupted, furin cleavage motif and/orfurin cleavage site at the carboxy-terminus of a Shiga toxin A1 fragmentderived region. In certain further embodiments, the Shiga toxin effectorpolypeptide does not comprise any known compensatory structure which mayprovide furin cleavage proximal to the carboxy-terminus of the Shigatoxin A1 fragment derived region. Non-limiting examples of disruptedfurin cleavage motifs and furin cleave sites suitable for use in thepresent invention are described in WO 2015/191764.

Certain furin-cleavage motif disruptions are indicated herein byreference to specific amino acid positions of native Shiga toxin ASubunits provided in the Sequence Listing, noting that naturallyoccurring Shiga toxin A Subunits includes precursor forms containingsignal sequences of about 22 amino acids at their amino-terminals whichare removed to produce mature Shiga toxin A Subunits and arerecognizable to the skilled worker. Further, certain furin-cleavagemotif disruptions comprising mutations are indicated herein by referenceto specific amino acids (e.g. R for an arginine residue) nativelypresent at specific positions within native Shiga toxin A Subunits (e.g.R251 for the arginine residue at position 251 from the amino-terminus)followed by the amino acid with which that residue has been substitutedin the particular mutation under discussion (e.g. R251A represents theamino acid substitution of alanine for arginine at amino acid residue251 from the amino-terminus).

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region, and suchembodiments are referred to herein as “furin-cleavage resistant” or“protease-cleavage resistant,” Shiga toxin effector polypeptides todescribe their property(ies) relative to wild-type, Shiga toxin ASubunits and/or wild-type, Shiga toxin A1 fragment fusion proteins.

In certain embodiments, the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention consists essentially of atruncated Shiga toxin A Subunit having two or more mutations.

In certain embodiments, the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention comprises the disruptedfurin-cleavage motif comprising the amino acid residue substitution(relative to a wild-type Shiga toxin polypeptide) of one or both of thearginine residues in the minimal, furin-cleavage site consensus motifwith A, G, or H. In certain embodiments, the protease-cleavageresistant, Shiga toxin effector polypeptide of the present inventioncomprises a disruption which comprises an amino acid substitution withina furin-cleavage motif region, where in the substitution occurs at thenatively positioned amino acid selected from the group consisting of:247 of SEQ ID NO:3, 248 of SEQ ID NO:1 or SEQ ID NO:2, 250 of SEQ IDNO:3, 251 of SEQ ID NO:1 or SEQ ID NO:2, or the equivalent position in aconserved Shiga toxin effector polypeptide and/or non-native Shiga toxineffector polypeptide sequence. In certain further embodiments, thesubstitution is to any non-conservative amino acid and the substitutionoccurs at the natively positioned amino acid residue position. Incertain further embodiments, the mutation comprises an amino acidsubstitution selected from the group consisting of: R247A, R248A, R250AR251A, or the equivalent position in a conserved Shiga toxin effectorpolypeptide and/or non-native Shiga toxin effector polypeptide sequence.

In certain embodiments, the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention comprises the disruptedfurin-cleavage motif comprising the mutation which is a deletion. Incertain further embodiments, the disrupted furin-cleavage motifcomprises a mutation which is a deletion of the region nativelypositioned at 247-252 in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO:3), orthe region natively positioned at 246-251 in SLT-2A (SEQ ID NO:3); adeletion of the region natively positioned at 244-246 in StxA (SEQ IDNO:2) and SLT-1A (SEQ ID NO:3), or the region natively positioned at243-245 in SLT-2A (SEQ ID NO:3); or a deletion of the region nativelypositioned at 253-259 in StxA (SEQ ID NO:2) and SLT-1A (SEQ ID NO:3), orthe region natively positioned at 252-258 in SLT-2A (SEQ ID NO:3).

In certain embodiments of the protease-cleavage resistant, Shiga toxineffector polypeptide of the present invention, the disruptedfurin-cleavage motif comprises the mutation which is a carboxy-terminaltruncation as compared to a wild-type Shiga toxin A Subunit, thetruncation which results in the deletion of one or more amino acidresidues within the furin-cleavage motif. In certain furtherembodiments, the disrupted furin-cleavage motif comprises thecarboxy-terminal truncation which deletes one or more amino acidresidues within the minimal cleavage site Y/R-x-x-R, such as, e.g., forStxA and SLT-1A derived Shiga toxin effector polypeptides, truncationsending at the natively amino acid residue position 250, 249, 248, 247,246, 245, 244, 243, 242, 241, 240, or less; and for SLT-2A derived Shigatoxin effector polypeptides, truncations ending at the natively aminoacid residue position 249, 248, 247, 246, 245, 244, 243, 242, 241, orless. Certain further embodiments comprise the disrupted furin-cleavagemotif comprising a combination of any of the aforementioned mutations,where possible.

In certain embodiments, the disrupted furin-cleavage motif comprises themutation(s) that is a partial, carboxy-terminal truncation of thefurin-cleavage motif, however, certain molecules of the presentinvention do not comprise the disrupted furin-cleavage motif which is acomplete, carboxy-terminal truncation of the entire 20 amino acidresidue, furin-cleavage motif. For example, certain, Shiga toxineffector polypeptides of the present invention comprise the disruptedfurin-cleavage motif comprising a partial, carboxy-terminal truncationof the Shiga toxin A1 fragment region up to native position 240 in StxA(SEQ ID NO:2) or SLT-1A (SEQ ID NO:1) but not a carboxy-terminaltruncation at position 239 or less. Similarly, certain, certain, Shigatoxin effector polypeptides of the present invention comprise thedisrupted furin-cleavage motif comprising a partial, carboxy-terminaltruncation of the Shiga toxin A1 fragment region up to native position239 in SLT-2A (SEQ ID NO:3) but not a carboxy-terminal truncation atposition 238 or less. In the largest carboxy-terminal truncation of thefurin-cleavage resistant, Shiga toxin effector polypeptide of thepresent invention, mutations comprising the disrupted furin-cleavagemotif, positions P14 and P13 of the furin-cleavage motif are stillpresent.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth an amino acid residue substitution within the furin-cleavage motifand a carboxy-terminal truncation as compared to a wild-type, Shigatoxin A Subunit. In certain further embodiments, the disruptedfurin-cleavage motif comprises both an amino acid residue substitutionwithin the minimal furin-cleavage site R/Y-x-x-R and a carboxy-terminaltruncation as compared to a wild-type, Shiga toxin A Subunit, such as,e.g., for StxA and SLT-1A derived Shiga toxin effector polypeptides,truncations ending at the natively amino acid residue position 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278,279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, orgreater and comprising the natively positioned amino acid residue R248and/or R251 substituted with any non-positively charged, amino acidresidue where appropriate; and for SLT-2A derived Shiga toxin effectorpolypeptides, truncations ending at the natively amino acid residueposition 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, or greater and comprising the natively positionedamino acid residue Y247 and/or R250 substituted with any non-positivelycharged, amino acid residue where appropriate. In certain embodiments,the truncated Shiga toxin effector polypeptide comprising a disruptedfurin-cleavage motif also comprises the furin-cleavage motif, amino acidresidues at positions P9, P8, and/or P7 in order to maintain optimalcytotoxicity.

In certain embodiments, the disrupted furin-cleavage motif comprises amutation(s) which is one or more internal, amino acid residue deletions,as compared to a wild-type, Shiga toxin A Subunit. In certain furtherembodiments, the disrupted furin-cleavage motif comprises a mutation(s)which has one or more amino acid residue deletions within the minimalfurin-cleavage site R/Y-x-x-R. For example, StxA and SLT-1A derivedShiga toxin effector polypeptides comprising internal deletions of thenatively positioned amino acid residues R248 and/or R251, which may becombined with deletions of surrounding residues such as, e.g., 249, 250,247, 252, etc.; and SLT-2A derived Shiga toxin effector polypeptidescomprising internal deletions of the natively positioned amino acidresidues Y247 and/or R250, which may be combined with deletions ofsurrounding residues such as, e.g., 248, 249, 246, 251, etc. In certainfurther embodiments, the disrupted furin-cleavage motif comprises amutation which is a deletion of four, consecutive, amino acid residueswhich deletes the minimal furin-cleavage site R/Y-x-x-R, such as, e.g.,StxA and SLT-1A derived Shiga toxin effector polypeptides lackingR248-R251 and SLT-2A derived Shiga toxin effector polypeptides lackingY247-R250. In certain further embodiments, the disrupted furin-cleavagemotif comprises a mutation(s) having one or more amino acid residuedeletions in the amino acid residues flanking the core furin-cleavagemotif, such as, e.g., a deletion of 244-247 and/or 252-255 in SLT-1A orStxA. In certain further embodiments, the disrupted furin-cleavage motifcomprises a mutation which is an internal deletion of the entiresurface-exposed, protease-cleavage sensitive loop as compared to awild-type, Shiga toxin A Subunit, such as, e.g., for StxA and SLT-1Aderived Shiga toxin effector polypeptides, a deletion of nativelypositioned amino acid residues 241-262; and for SLT-2A derived Shigatoxin effector polypeptides, a deletion of natively positioned aminoacid residues 240-261.

In certain embodiments, the disrupted furin-cleavage motif comprisesboth a mutation which is an internal, amino acid residue deletion withinthe furin-cleavage motif and a mutation which is carboxy-terminaltruncation as compared to a wild-type, Shiga toxin A Subunit. In certainfurther embodiments, the disrupted furin-cleavage motif comprises both amutation which is an amino acid residue deletion within the minimalfurin-cleavage site R/Y-x-x-R and a mutation which is a carboxy-terminaltruncation as compared to a wild-type, Shiga toxin A Subunit. Forexample, protease-cleavage resistant, Shiga toxin effector polypeptidesmay comprise a disrupted furin-cleavage motif comprising mutation(s)which are deletions of the natively positioned amino acid residues248-249 and/or 250-251 in a truncated StxA or SLT-1A polypeptide whichstill has amino acid residue 247 and/or 252, or the amino acid residues247-248 and/or 249-250 in a truncated SLT-2A which still has amino acidresidue 246 and/or 251. In certain further embodiments, the disruptedfurin-cleavage motif comprises a mutation having a deletion of four,consecutive, amino acid residues which deletes the minimalfurin-cleavage site R/Y-x-x-R and a carboxy-terminal truncation ascompared to a wild-type, Shiga toxin A Subunit, such as, e.g., for StxAand SLT-1A derived Shiga toxin effector polypeptides, truncations endingat the natively amino acid residue position 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, or greater and lacking R248-R251; andfor SLT-2A derived Shiga toxin effector polypeptides, truncations endingat the natively amino acid residue position 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, or greater and lackingY247-R250.

C. Examples of Shiga Toxin Effector Polypeptides Having an EmbeddedEpitope

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention may comprise one or more embedded or inserted,heterologous, T-cell epitopes for purposes of de-immunization and/ordelivery to a MHC class I presentation pathway of a target cell. Forcertain embodiments and/or certain Shiga toxin effector polypeptidesub-regions, embedding or partial embedding a T-cell epitope may bepreferred over inserting a T-cell epitope because, e.g., embedding-typemodifications are more likely to be successful in diverse sub-regions ofa Shiga toxin effector polypeptide whereas successful insertions may bemore limited to a smaller subset of Shiga toxin effector polypeptidesub-regions. The term “successful” is used here to mean the modificationto the Shiga toxin effector polypeptide (e.g. introduction of aheterologous, T-cell epitope) results in a modified Shiga toxin effectorpolypeptide which retains one or more Shiga toxin effector functions atthe requisite level of activity either alone or as a component of acell-targeting molecule.

Any of the Shiga toxin effector polypeptide sub-regions described in WO2015/113007 may be suitable for certain embodiments of the presentinvention, and any of the Shiga toxin effector polypeptides described inWO 2015/113007 may be modified into a Shiga toxin effector polypeptideof the present invention, e.g., by the addition of one or more newepitope region disruptions for de-immunization (such one as describedherein) and/or a furin-cleavage motif disruption (such as one describedherein).

In certain embodiments, the Shiga toxin effector polypeptide of thepresent invention consists essentially of a truncated Shiga toxin ASubunit comprising an embedded or inserted, heterologous, T-cell epitopeand one or more other mutations. In certain embodiments, the Shiga toxineffector polypeptide of the present invention comprises an embedded orinserted, heterologous, T-cell epitope and is smaller than afull-length, Shiga toxin A Subunit, such as, e.g., consisting of thepolypeptide represent by amino acids 77 to 239 of SLT-1A (SEQ ID NO:1)or StxA (SEQ ID NO:2) or the equivalent in other A Subunits of membersof the Shiga toxin family (e.g. amino acids 77 to 238 of SLT-2A (SEQ IDNO:3)). For example, in certain embodiments of the present invention,the Shiga toxin effector polypeptides is derived from amino acids 75 to251 of SEQ ID NO:1, 1 to 241 of SEQ ID NO:1, 1 to 251 of SEQ ID NO:1, oramino acids 1 to 261 of SEQ ID NO:1, wherein the Shiga toxin effectorpolypeptide comprises at least one embedded or inserted, heterologousT-cell epitope and at least one amino acid is disrupted in anendogenous, B-cell and/or CD4+ T-cell epitope region provided in theExamples (see e.g. Tables 1-7 and/or 12) and wherein the disrupted aminoacid does not overlap with the embedded or inserted epitope. Similarlyin other embodiments, the Shiga toxin effector polypeptide of thepresent invention is derived from amino acids 75 to 251 of SEQ ID NO:2,1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or amino acids 1 to261 of SEQ ID NO:2, wherein the Shiga toxin effector polypeptidecomprises at least one embedded or inserted, heterologous T-cell epitopeand at least one amino acid is disrupted in an endogenous, B-cell and/orCD4+ T-cell epitope region provided in the Examples (see e.g. Tables 1-7and/or 12) and wherein the disrupted amino acid does not overlap withthe embedded or inserted epitope. Additionally, the Shiga toxin effectorpolypeptide may be derived from amino acids 75 to 251 of SEQ ID NO:3, 1to 241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3, or amino acids 1 to 261of SEQ ID NO:3, wherein the Shiga toxin effector polypeptide comprisesat least one embedded or inserted, heterologous T-cell epitope and atleast one amino acid is disrupted in an endogenous, B-cell and/or CD4+T-cell epitope region provided in the Examples (see e.g. Tables 1-7and/or 12) and wherein the disrupted amino acid does not overlap withthe embedded or inserted epitope. In certain embodiments of the presentinvention, the Shiga toxin effector polypeptide comprises an embedded orinserted, heterologous, T-cell epitope and a disrupted furin-cleavagemotif at the carboxy-terminus of a Shiga toxin A1 fragment derivedregion. For example in certain embodiments, the Shiga toxin effectorpolypeptide of the present invention is derived from amino acids 75 to251 of SEQ ID NO:1, 1 to 241 of SEQ ID NO:1, 1 to 251 of SEQ ID NO:1, oramino acids 1 to 261 of SEQ ID NO:1, wherein the Shiga toxin effectorpolypeptide comprises at least one embedded or inserted, heterologousT-cell epitope and a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region. Similarlyin other embodiments, the Shiga toxin effector polypeptide of thepresent invention is derived from amino acids 75 to 251 of SEQ ID NO:2,1 to 241 of SEQ ID NO:2, 1 to 251 of SEQ ID NO:2, or amino acids 1 to261 of SEQ ID NO:2, wherein the Shiga toxin effector polypeptidecomprises at least one embedded or inserted, heterologous T-cell epitopeand a disrupted furin-cleavage motif at the carboxy-terminus of a Shigatoxin A1 fragment derived region. Additionally, the Shiga toxin effectorpolypeptide may be derived from amino acids 75 to 251 of SEQ ID NO:3, 1to 241 of SEQ ID NO:3, 1 to 251 of SEQ ID NO:3, or amino acids 1 to 261of SEQ ID NO:3, wherein the Shiga toxin effector polypeptide comprisesat least one embedded or inserted, heterologous T-cell epitope and adisrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxinA1 fragment derived region.

D. Examples of Combination Shiga Toxin Effector Polypeptides

A combination Shiga toxin effector polypeptide of the present inventioncomprises two or more sub-regions (i.e. non-overlapping sub-regions)wherein each sub-region comprises at least one of the following: (1) adisruption in an endogenous epitope or epitope region; (2) an embedded,heterologous, T-cell epitope-peptide; (3) an inserted, heterologous,T-cell epitope-peptide; and (4) a disrupted furin-cleavage motif at thecarboxy-terminus of an A1 fragment derived region.

Certain embodiments of the combination Shiga toxin effector polypeptidesof the present invention comprise both (1) a disruption in an endogenousepitope or epitope region and (2) a disrupted furin-cleavage motif atthe carboxy-terminus of an A1 fragment derived region. It is predictedthat any of the individual, de-immunized, Shiga toxin effectorsub-regions described in the Examples below or described in WO2015/113005 (see e.g. Table B, supra) may generally be combined with anyShiga toxin effector sub-region comprising a disrupted furin-cleavagemotif described herein, described in WO 2015/191764, and/or known in theart in order to create a Shiga toxin effector polypeptide of the presentinvention.

In certain embodiments of the present invention, the Shiga toxineffector polypeptide consists essentially of the polypeptide shown inany one of SEQ ID NOs: 355-438 which further comprises a disruption ofat least one, endogenous, B-cell and/or T-cell epitope region which doesnot overlap with an embedded or inserted, heterologous, CD8+ T-cellepitope; wherein the disruption comprises one or more amino acid residuesubstitutions relative to a wild-type Shiga toxin. In certain furtherembodiments the substitution is selected from the group consisting of:K1 to A, G, V, L, I, F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 toA, G, V, L, I, F, S, Q and R; S8 to A, G, V, I, L, F, and M; T9 to A, G,V, I, L, F, M, and S; S9 to A, G, V, L, I, F, and M; K11 to A, G, V, L,I, F, M and H; T12 to A, G, V, I, L, F, M, S, and K; S12 to A, G, V, I,L, F, and M; S33 to A, G, V, L, I, F, M, and C; S43 to A, G, V, L, I, F,and M; G44 to A or L; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L,I, F, and M; G46 to A and P; D47 to A, G, V, L, I, F, S, M, and Q; N48to A, G, V, L, M and F; L49 to A, V, C, and G; Y49 to A, G, V, L, I, F,M, and T; F50 to A, G, V, L, I, and T; A51; D53to A, G, V, L, I, F, S,and Q; V54 to A, G, I, and L; R55 to A, G, V, L, I, F, M, Q, S, K, andH; G56 to A and P; 157 to A, G, V, and M; L57 to A, V, C, G, M, and F;D58 to A, G, V, L, I, F, S, and Q; P59 to A, G, and F; E60 to A, G, V,L, I, F, S, Q, N, D, M, T, and R; E61 to A, G, V, L, I, F, S, Q, N, D,M, and R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to Aand G; I88 to A, V, C, and G; D94 to A, G, V, L, I, F, S, and Q; S96 toA, G, V, I, L, F, and M; T104 to A, G, V, L, I, F, M; and N; A105 to L;T107 to A, G, V, L, I, F, M, and P; S107 to A, G, V, L, I, F, M, and P;L108 to A, V, C, and G; S109 to A, G, V, I, L, F, and M; T109 to A, G,V, I, L, F, M, and S; G110 to A; S112 to A, G, V, L, I, F, and M; D111to A, G, V, L, I, F, S, Q, and T; S112 to A, G, V, L, I, F, and M; D141to A, G, V, L, I, F, S, and Q; G147 to A; V154 to A and G. R179 to A, G,V, L, I, F, M, Q, S, K, and H; T180 to A, G, V, L, I, F, M, and S; T181to A, G, V, L, I, F, M, and S; D183 to A, G, V, L, I, F, S, and Q; D184to A, G, V, L, I, F, S, and Q; L185 to A, G, V and C; S186 to A, G, V,I, L, F, and M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, and H;S189 to A, G, V, I, L, F, and M; D198 to A, G, V, L, I, F, S, and Q;R204 to A, G, V, L, I, F, M, Q, S, K, and H; R205 to A, G, V, L, I, F,M, Q, S, K and H; S247 to A, G, V, I, L, F, and M; Y247 to A, G, V, L,I, F, and M; R248 to A, G, V, L, I, F, M, Q, S, K, and H; R250 to A, G,V, L, I, F, M, Q, S, K, and H; R251 to A, G, V, L, I, F, M, Q, S, K, andH; D264 to A, G, V, L, I, F, S, and Q; G264 to A; and T286 to A, G, V,L, I, F, M, and S. In certain further embodiments, there are multipledisruptions of multiple, endogenous B-cell and/or CD8+ T-cell epitoperegions wherein each disruption involves at least one amino acid residuesubstitution selected from the group consisting of: K1 to A, G, V, L, I,F, M and H; T4 to A, G, V, L, I, F, M, and S; D6 to A, G, V, L, I, F, S,Q and R; S8 to A, G, V, I, L, F, and M; T9 to A, G, V, I, L, F, M, andS; S9 to A, G, V, L, I, F, and M; K11 to A, G, V, L, I, F, M and H; T12to A, G, V, I, L, F, M, S, and K; S12 to A, G, V, I, L, F, and M; S33 toA, G, V, L, I, F, M, and C; S43 to A, G, V, L, I, F, and M; G44 to A orL; S45 to A, G, V, L, I, F, and M; T45 to A, G, V, L, I, F, and M; G46to A and P; D47 to A, G, V, L, I, F, S, M, and Q; N48 to A, G, V, L, Mand F; L49 to A, V, C, and G; Y49 to A, G, V, L, I, F, M, and T; F50 toA, G, V, L, I, and T; A51; D53 to A, G, V, L, I, F, S, and Q; V54 to A,G, I, and L; R55 to A, G, V, L, I, F, M, Q, S, K, and H; G56 to A and P;157 to A, G, V, and M; L57 to A, V, C, G, M, and F; D58 to A, G, V, L,I, F, S, and Q; P59 to A, G, and F; E60 to A, G, V, L, I, F, S, Q, N, D,M, T, and R; E61 to A, G, V, L, I, F, S, Q, N, D, M, and R; G62 to A;R84 to A, G, V, L, I, F, M, Q, S, K, and H; V88 to A and G; 188 to A, V,C, and G; D94 to A, G, V, L, I, F, S, and Q; S96 to A, G, V, I, L, F,and M; T104 to A, G, V, L, I, F, M; and N; A105 to L; T107 to A, G, V,L, I, F, M, and P; S107 to A, G, V, L, I, F, M, and P; L108 to A, V, C,and G; S109 to A, G, V, I, L, F, and M; T109 to A, G, V, I, L, F, M, andS; G110 to A; S112 to A, G, V, L, I, F, and M; D111 to A, G, V, L, I, F,S, Q, and T; S112 to A, G, V, L, I, F, and M; D141 to A, G, V, L, I, F,S, and Q; G147 to A; V154 to A and G. R179 to A, G, V, L, I, F, M, Q, S,K, and H; T180 to A, G, V, L, I, F, M, and S; T181 to A, G, V, L, I, F,M, and S; D183 to A, G, V, L, I, F, S, and Q; D184 to A, G, V, L, I, F,S, and Q; L185 to A, G, V and C; S186 to A, G, V, I, L, F, and M; G187to A; R188 to A, G, V, L, I, F, M, Q, S, K, and H; S189 to A, G, V, I,L, F, and M; D198 to A, G, V, L, I, F, S, and Q; R204 to A, G, V, L, I,F, M, Q, S, K, and H; R205 to A, G, V, L, I, F, M, Q, S, K and H; S247to A, G, V, I, L, F, and M; Y247 to A, G, V, L, I, F, and M; R248 to A,G, V, L, I, F, M, Q, S, K, and H; R250 to A, G, V, L, I, F, M, Q, S, K,and H; R251 to A, G, V, L, I, F, M, Q, S, K, and H; D264 to A, G, V, L,I, F, S, and Q; G264 to A; and T286 to A, G, V, L, I, F, M, and S.

Certain embodiments of the Shiga toxin effector polypeptides of thepresent invention comprise both (1) an embedded or inserted,heterologous, T-cell epitope-peptide and (2) a disrupted furin-cleavagemotif at the carboxy-terminus of an A1 fragment derived region. Any ofthe Shiga toxin effector polypeptide sub-regions comprising an embeddedor inserted, heterologous, T-cell epitope described in the Examplesbelow or in WO 2015/113007 may generally be combined with anyprotease-cleavage resistant, Shiga toxin effector polypeptide sub-region(e.g., modified, Shiga toxin A Subunit sub-regions described herein,described in WO 2015/191764, and/or known in the art) in order to createa combination, Shiga toxin effector polypeptide which, as a component ofa cell-targeting molecule, is both protease-cleavage resistant andcapable of delivering a heterologous, T-cell epitope to the MHC class Ipresentation pathway of a target cell. Non-limiting examples of thistype of combination Shiga toxin effector polypeptide are shown in SEQ IDNOs: 6-27, 29-32, 340-355, and 370-438.

Certain embodiments of the combination Shiga toxin effector polypeptidesof the present invention comprise both (1) a disruption in an endogenousepitope or epitope region and (2) an embedded, heterologous, T-cellepitope-peptide. However, the Shiga toxin effector sub-regionscomprising inserted or embedded, heterologous, T-cell epitopes describedherein or in WO 2015/191764 are generally not combinable with everyde-immunized, Shiga toxin effector sub-regions described herein, exceptwhere empirically shown to be successfully combined such that theresulting combination molecule retained a sufficient level of a Shigatoxin effector function(s). The disclosure herein shows how suchembodiments may be made and tested to empirically demonstrate success.

The term “successful” is used here to mean two or more amino acidresidue substitutions in a Shiga toxin effector polypeptide results in afunctional feature, such as, e.g., de-immunization, reducedfurin-cleavage, and/or ability to deliver an embedded or insertedepitope, while the modified Shiga toxin effector polypeptide retains oneor more Shiga toxin effector functions. The approaches and assaysdescribed herein show how to design, make and empirically testembodiments of the present invention, which represent combination, Shigatoxin effector polypeptides and cell-targeting molecules comprising thesame.

The combination, Shiga toxin effector polypeptides of the presentinvention combine the features of their respective sub-regions, such as,e.g., a furin-cleavage motif disruption, individual epitope disruptions,and/or a heterologous T-cell epitope cargo, and these combinationssometimes result in Shiga toxin effector polypeptides with synergisticreductions in immunogenicity as compared to the sum of their partiallyde-immunized sub-regions. In particular, the exemplary, Shiga toxineffector polypeptides shown in SEQ ID NOs: 13, 16 and 21 aresynergistically de-immunized due to the combination of two or moresub-regions, one of which comprises an embedded, heterologous, T-cellepitope and another of which comprises a endogenous epitope disrupted byone or more amino acid residue substitutions.

For certain embodiments, the Shiga toxin effector polypeptide of thepresent invention comprises or consists essentially of the polypeptideshown in any one of SEQ ID NOs: 6-32, 340-354, and 370-438. For certainembodiments, the combination, de-immunized, protease-cleavage resistant,Shiga toxin effector polypeptides comprising embedded, T-cell epitopesof the present invention comprise or consist essentially of one of thepolypeptides represented by SEQ ID NOs: 6-10, 13-32, 340-354, and370-438.

De-immunized, Shiga toxin effector polypeptides of the present inventionwhich exhibit no cytotoxicity or reduced cytotoxicity at certainconcentrations, e.g. Shiga toxin effector polypeptides comprising R179A,may still be useful as de-immunized, Shiga toxin effector polypeptidesfor delivering exogenous materials into cells. Similarly, CD8+ T-cellhyper-immunized, Shiga toxin effector polypeptides of the presentinvention which exhibit no cytotoxicity or reduced cytotoxicity atcertain concentrations, e.g. a Shiga toxin effector polypeptidecomprising an epitope embedded into its catalytic domain (see e.g. WO2015/113007, Example 1-F), may still be useful for delivering a T-cellepitope(s) to a desired subcellular compartment of a cell in which theShiga toxin effector polypeptide is present or as a component of acell-targeting molecule for delivery of a T-cell epitope(s) into atarget cell.

E. Examples of Cell-Targeting Molecules of the Present Invention

The Shiga toxin effector polypeptides of the present invention may beused as components of cell-targeting molecules that target variousextracellular target biomolecules. The following examples describe inmore detail certain structures of exemplary cell-target molecules of thepresent invention which target cells expressing extracellular targetbiomolecules such as, e.g., CD19, CD20, CD22, CD30, CD38, CD45, HER2,PD-L1, and TYRP1.

1. Cell-Targeting Molecules Targeting Human CD19

CD19, also recognized in the art as B4, is a 95 kDa, B-lineage specific,type-I transmembrane glycoprotein present on the surface of developingB-cells but not expressed by terminally differentiated plasma cells.While the name CD19 might refer to multiple proteins with relatedstructures and polypeptide sequences from various species, for thepurposes of the structural examples of this section, the term “CD19”refers to the B-lymphocyte antigen CD19 proteins present in humans whoseexact sequence might vary slightly based on the isoform and fromindividual to individual. With regard to humans, CD19 refers to theprotein represented by the predominant polypeptide sequence UniProtP15391 and (National Center Biotechnology Institute, U.S.) (NCBI)accession AAA69966.1 or AAB60697.1; however, different isoforms andvariants exist due to splicing, polymorphisms and/or mutations (seee.g., Kuroki K et al., Genes Immun Suppl 1: S21-30 (2002); Tsuchiya N etal., Arthritis Rheum 50: 4002-7 (2004); Dawidowicz K et al., Clin ExpRheumatol 29: 839-42 (2011)). A skilled worker will be able to identifyother CD19 proteins in humans, even if they differ from the referencedsequences.

CD19 is an attractive target for targeted cancer therapies, e.g.,because of the ubiquitous cell-surface expression of CD19 by neoplasticcells and tumors of B-cell lineages. For example, most malignant B-cellswere found to express CD19 (see e.g., Anderson K et al., Blood 63: 1424(1984); Uckun F et al., Blood 71: 13 (1988); Bradbury L et al., JImmunol 149: 2841-50 (1992); Haas K, Tedder T, Adv Exp Med Biol 560:125-39 (2005); Tedder T, Nat Rev Rheumatol 5: 572-7 (2009)). AlthoughCD19 is considered a pan B-cell marker expressed throughout B-celldevelopment, mature B-cells and tumor cells of B-cell lineages have beenobserved to express three-fold more CD19 compared to immature B-cells.In particular, CD19 expression was observed in indolent and aggressivesubtypes of non-Hodgkin lymphoma (NHL), B-cell chronic lympocyticleukemia (B-CLL), and forms of acute lymphoblastic leukemia.Furthermore, due to differences in CD19 expression as compared to CD20expression, CD19-targeted therapies may be able to target B-cellneoplasms at early stages than CD20-targeted therapies.

There are numerous CD19 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “CD19 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a CD19molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD20 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, CD19 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human CD19.

In certain embodiments, the CD19 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, CD19 binding region is derived from animmunoglobulin, CD19 binding region, such as an antibody paratopecapable of binding an extracellular part of CD19. In certain otherembodiments, the immunoglobulin-type, CD19 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, CD19 binding region by providinghigh-affinity binding to an extracellular part of CD19. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous CD19 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,CD19 binding regions include CD19-binding monoclonal antibodies andderivatives thereof, such as humanized variants and recombinantimmunoglobulin domains, e.g., B4 (e.g. clone eBiolD3), Leu-12 (Leu12),HD37, B43, CLB-CD19, MOPC 21 components, FMC63, MB19-1, cCD19, B4 89B,SJ25-C1, hA19, huB4, hBU12, XmAb5574, MOR208, MEDI-551, SAR3419, AFM11,GBR 401, XmAb 5871, Hm2E8b, B-1, 5F3, 2E2, 1G9, C-20, F-3, HD237, H-300,M-20, R-20, PDR134, BCE19, HIB19, LE-CD19, LT19, CB19, 6D5, 4G7, AB-1,F974A2, J3-119, MDX-1342, MAB7489 (clone 771404), and MAB4867 (clone4G7-2E3) (see e.g., Caligaris-Cappio F et al., J Clin Invest 76: 1243-51(1985); Chen Z et al., Leuk Res 10: 1411-7 (1986); Pezzutto A et al., JImmunol 138: 2793-9 (1987); De Rie M et al., Leuk Res 12: 135-41 (1988);Uckun F et al., Blood 71: 13-29 (1988); Vuist W et al., Cancer Res 49:3783-8 (1989); Carter R et al., J Immunol 147: 3663-71 (1991); Zola H etal., Immunol Cell Biol 69: 411-22 (1991); Holder M et al., Eur J Immunol22: 2725-8 (1992); Engel P et al., Immunity 3: 39-50 (1995); Pietersz Get al., Cancer Immunol Immunother 41: 53-60 (1995); Tisone J et al., AmJ Clin Pathol 107: 283-91 (1997); WO 2005/012493; Lutz R et al., Proc AmAssoc Cancer Res 47: 3731 (2006); Horton H et al., Cancer Res 68:8049-57 (2008); Gerber H et al., Blood 113: 4352-61 (2009); Awan F etal., Blood 115: 1204-13 (2010); Herbst R et al., J Pharmacol Exp Ther335: 213-22 (2010); Coiffier B et al., J Clin Oncol 29: 1182-9 (2011);Reusch U et al., Blood 122: 4405 (2013); Breton C et al., J HematolOncol 7: 33 (2014); Horton H et al., J Immunol 186: 4223-33 (2014); ShenD et al., Monoclon Antib Immunodiagn Immunother 33: 215-20 (2014)).Non-limiting examples of CD19 binding regions include scFvs, such as,e.g., FVS191, FVS192, scFv-HD37, scFv-FMC63, HD37-C, HD37-CCH,FMC63-28Z, 4G7mut, 4G7-graft (see e.g., Bejeck B et al., Cancer Res 55:2346-51 (1995); Kipriyanov et al., J Immunol Meth 196: 51-62 (1996);Nicholson I et al., Mol Immunol 34: 1157-65 (1997); WO 2002/050118;Peipp M et al., J Immunol Methods 285: 265-80 (2004); Cheng W et al.,Biochim Biophys Acta 1768: 21-9 (2007); Kochenderfer J et al., JImmunother 32: 689-702 (2009); Kugler M et al., Protein Eng Des Sel 22:135-47 (2009); WO 2012/079000; Kneissi S et al., PLoS One 8: e79047(2013)).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanCD19 and/or the cellular surface of a CD19+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HABR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:83, SEQ ID NO:89, or SEQ ID NO:96; (ii) a HABR2comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:84, SEQ ID NO:90, SEQ ID NO:95, or SEQ ID NO:97;and (iii) a HABR3 comprising or consisting essentially of one of theamino acid sequence as shown in SEQ ID NO:85, SEQ ID NO:91, or SEQ IDNO:98; and b) a light chain variable (V_(L)) domain comprising (i) aLABR1 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:86, SEQ ID NO:92, or SEQ ID NO:99; (ii) aLABR2 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:97, SEQ ID NO:93, or SEQ ID NO:100; and(iii) a LABR3 comprising or consisting essentially of one of the aminoacid sequence as shown in SEQ ID NO:88, SEQ ID NO:94, or SEQ ID NO:101.In certain further embodiments, the cell-targeting molecule of thepresent invention comprises the binding region comprising or consistingessentially of amino acids 1-232, 1-233, 1-234, 1-235, 1-236, 1-242,1-243, 1-244, 1-245, 1-246, 1-252, 1-253, 1-254, 1-255, or 1-256 of anyone of SEQ ID NOs: 47-119 and 176-248.

According to one specific but non-limiting aspect, the binding region ofthe cell-targeting molecule of the present invention comprises a ligand(whether naturally occurring or synthetic) or a derivative thereof thatretains binding functionality to an extracellular part of CD19. NativeCD19 is known to bind at least one ligand, CD19-L, a high mobility group(HMG) box protein (see e.g., Uckun F et al., Br J Haematol 153: 15-23(2011); US 20120141505).

Any of the aforementioned CD19 binding molecules may be suitable for useas a CD19 binding region or modified to create one or more CD19 bindingregions for use in a cell-targeting molecule of the present invention.

2. Cell-Targeting Molecules Targeting Human CD20

CD20 (B-lymphocyte antigen CD20) While the name CD20 might refer tomultiple proteins with related structures and polypeptide sequences fromvarious species, for the purposes of the structural examples of thissection, the term “CD20” refers to the B-lymphocyte antigen CD20proteins present in humans whose exact sequence might vary slightlybased on the isoform and from individual to individual. With regard tohumans, CD20 refers to the protein represented by the predominantpolypeptide sequence UnitProt P11836 and NCBI accession NP 690605.1;however, different isoforms and variants exist due to splicing,polymorphisms and/or mutations (see e.g., Dawidowicz K et al., Clin ExpRheumatol 29: 839-42 (2011); Fang C et al., Int J Clin Exp Med 8:11235-43 (2015)). A skilled worker will be able to identify other CD20proteins in humans, even if they differ from the referenced sequences.

CD20 is a cell-surface glycoprotein expressed by normal, B-cell lineagecells within certain cell developmental stages as well as cells ofnumerous, mature B-cell neoplasms, such as NHL and chronic lymphocyticleukemia (CLL) cells. In addition, CD20 is expressed by mature T-celland NK-cell neoplasms. CD20 is expressed by a subset of normal T-cellsas well as malignant T-cells such as, e.g., in T-cell lymphomas (TCLs)including mycosis fungoides (MF), natural killer cell lymphoma (NK-celllymphoma), peripheral T-cell lymphomas (PTCLs), cutaneous T-celllymphomas, and T-cell large granular lymphocyte leukemia (T-LGLL). Theassociation of cell-surface CD20 with malignant cells makes it anattractive target for cell-targeted therapies.

There are numerous CD20 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “CD20 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a CD20molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD20 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, CD20 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human CD20.

In certain embodiments, the CD20 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, CD20 binding region is derived from animmunoglobulin, CD20 binding region, such as an antibody paratopecapable of binding an extracellular part of CD20. In certain otherembodiments, the immunoglobulin-type, CD20 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, CD20 binding region by providinghigh-affinity binding to an extracellular part of CD20. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous CD20 binding regions contemplated as components ofthe present invention, such as, e.g. CD20 binding regions described inPCT/US2016/016580. Non-limiting examples of immunoglobulin-type, CD20binding regions include monoclonal antibodies and derivatives (e.g.,humanized variants and scFvs) such as, e.g., 1F5, 1H4, 1K1791, 2B8,Leu16, Leuδ, 2F2, 2H7, 7D8, 8E4, 11B8, AME-133v, LY2469298, B9E9, BM-ca,C2B8, CKI, GA101, R05072759, LT20, ibritumomab, HB20-1-25, MB20-1-18,obinutuzumab, ocaratuzumab, ocrelizumab, PRO70769, ofatumumab,OUBM1-OUBM8, PRO131921, rituximab, TGLA, tositumomab, TRU-015,ublituximab, veltuzumab, IMMU-106, hA20, the CD20-binding fibronectindomain FN3CD20, and HL23-scFvs: scFv-1, scFv-3, scFv-5, and scFv-8 (seee.g. Golay J et al., J Immunol 135: 3795-801 (1985); Tedder T et al.,Eur J Immunol 16: 881-7 (1986); Liu A et al., Proc Natl Acad Sci USA 84:3439-43 (1987); Press 0 et al., Blood 69: 584-91 (1987); Maloney D etal., Blood 84: 2457-66 (1994); Reff M et al., Blood 83: 435-45 (1994);Hooijberg E et al., Cancer Res 55: 840-6 (1995); Hooijberg E et al.,Hybridoma 15: 23-31 (1996); Anderson D et al., Biochem Soc Trans 25:705-8 (1997); Haisma H et al., Blood 92: 184-90 (1998); Wiseman G etal., Clin Cancer Res 5: 3281s-3286s (1999); Schultz J et al., Cancer Res60: 6663-9 (2000); Cardarelli P et al., Cancer Immunol Immunother 51:15-24 (2002); Cheson B, Curr Opin Investig Drugs 3: 165-70 (2002);Polyak M et al., Blood 99: 3256-62 (2002); Teeling J et al., Blood 104:1793-800 (2004); Geng S et al., Cell Mol Immunol 3: 439-43 (2006); deBoer 0 et al., PLoS One 2: e779 (2007); Burge D et al., Clin Ther 30:1806-16 (2008); Hagenbeek A et al., Blood 111: 5486-95 (2008); Nishida Met al., Intl J Oncol 32: 1263-74 (2008); Morschhauser F et al., J ClinOncol 27: 3346-53 (2009); Lim S et al., Haematologica 95: 135-43 (2010);Lv M et al., Cancer Lett 294: 66-73 (2010); Morschhauser F et al., AnnOncol 21: 1870-6 (2010); Mossner E et al., Blood 115: 4393-402 (2010);Olafesn T et al., Protein Eng Des Sel 23: 243-9 (2010); Uchiyama S etal., Cancer Sci 101: 201-9 (2010); Wu L et al., Cancer Lett 292: 208-14(2010); Alduaij W et al., Blood 117: 4519-29 (2011); Boross P et al.,Haematologica 96: 1822-30 (2011); Fang H et al., Sci China Life Sci 54:255-62 (2011); Nickerson-Nutter C et al., Rheumatology 50: 1033-44(2011); Robak T, Robak E, BioDrugs 25: 13-25 (2011); Cang S et al., JHematol Oncol 5: 64 (2012); Salles G et al., Blood 119: 5126-32 (2012);Abdelwahed R et al., Invest Ophthalmol Vis Sci 54: 3657-65 (2013); GolayJ et al., Blood 122: 3482-91 (2013); Kinder M et al., J Biol Chem 288:3084-54 (2013); Kobayashi H et al., Cancer Med 2: 130-43 (2013);Natarajan A et al., Clin Cancer Res 19: 6820-9 (2013); Zhang H et al.,Cell Physiol Biochem 32: 645-54 (2013); Ahmadzadeh V et al., ProteinExpr Purif 102: 45-41 (2014); Ellbrecht C et al., JAMA Dermatol 1939(2014); Garff-Tavernier M et al., Leukemia 28: 230-3 (2014); U.S. Pat.Nos. 4,861,579; 5,500,362; 5,595,721; 5,677,180; 5,721,108; 5,736,137;5,776,456; 5,843,398; 5,849,898; 6,015,542; 6,090,365; 6,120,767;6,171,586; 6,194,551; 6,224,866; 6,242,195; 6,287,537; 6,306,393;6,368,596; 6,399,061; 6,410,391; 6,455,043; 6,528,624; 6,538,124;6,565,827; 6,652,852; 6,682,734; 7,879,984; 8,101,179; 8,153,125;8,337,844; and patent application publications WO 1995/03770; WO1998/58964; WO 1999/22764; WO 2000/09160; WO 2000/27428; WO 2000/27433;WO 2000/42072; WO 2000/44788; WO 2000/67795; WO 2000/67796; WO2000/76542; WO 2001/03734; WO 2001/10460; WO 2001/10461; WO 2001/10462;WO 2001/13945; WO 2001/72333; WO 2001/80884; WO 2001/97858; WO2002/060955; WO 2002/079255; WO 2002/096948; WO 2002/102312; WO2003/002607; WO 2003/061694; WO 2004/032828; WO 2005/000901; WO2005016969; WO 2006/106959; WO 2009/031230; WO 2014/076292; US2011/0091483; US 12/0941,583; PCT/US2010/055826; EP20140151932;PCT/GB2012/052532; U.S. Ser. No. 13/048,135; EP20140151932;PCT/GB2012/052532; U.S. Ser. No. 13/048,135; PCT/US2006/046034).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanCD20 and/or the cellular surface of a CD20+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HCDR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:102, SEQ ID NO:108, SEQ ID NO:114, SEQ ID NO:120,or SEQ ID NO:124; (ii) a HCDR2 comprising or consisting essentially ofone of the amino acid sequence as shown in SEQ ID NO:103, SEQ ID NO:115,or SEQ ID NO:125; and (iii) a HCDR3 comprising or consisting essentiallyof one of the amino acid sequence as shown in SEQ ID NO:104, SEQ IDNO:109, SEQ ID NO:111, SEQ ID NO:116, SEQ ID NO:121, or SEQ ID NO:126;and b) a light chain variable (V_(L)) domain comprising (i) a LCDR1comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:105, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:117,or SEQ ID NO:127; (ii) a LCDR2 comprising or consisting essentially ofone of the amino acid sequence as shown in SEQ ID NO:106, SEQ ID NO:118, SEQ ID NO:122, or SEQ ID NO:128; and (iii) a LCDR3 comprising orconsisting essentially of one of the amino acid sequence as shown SEQ IDNO:107, SEQ ID NO:113, SEQ ID NO:119, SEQ ID NO:123, or SEQ ID NO:129.In certain further embodiments, the binding region comprises or consistsessentially of amino acids 1-245 of any one of SEQ ID NOs: 33, 64, and65.

Any of the aforementioned CD20 binding molecules may be suitable for useas a CD20 binding region or modified to create one or more CD20 bindingregions for use in a cell-targeting molecule of the present invention.

3. Cell-Targeting Molecules Targeting Human CD22

CD22, also recognized in the art as Siglec-2, SIGLEC2, BL-CAM, B3,Leu-14, and Lyb-8, is a transmembrane glycoprotein of about 120-140 kDa(depending on the spliceoform) that binds sialic acid ligands. CD22 isexpressed specifically by B-cells during development and by a specificsubset of mature B-cells. While the name CD22 might refer to multipleproteins with related structures and polypeptide sequences from variousspecies, for the purposes of the structural examples of this section,the term “CD22” refers to sialic acid-binding lectin proteins present inhumans whose exact sequence might vary slightly based on the isoform andfrom individual to individual. With regard to humans, CD22 refers to theprotein represented by the predominant polypeptide sequence UniProtP20273 and NCBI accession NP_001265346.1; however, different isoformsand variants exist due to splicing, polymorphisms and/or mutations (seee.g., Hitomi Y et al., Tissue Antigens 69: 242-9 (2007); Dawidowicz K etal., Clin Exp Rheumatol 29: 839-42 (2011)). A skilled worker will beable to identify other CD22 proteins in humans, even if they differ fromthe referenced sequences.

As B-cell specific marker, CD22 is an attractive target forcell-targeted therapies for diseases and conditions involving B-cells,such as, e.g., conditions involving overactive B-cells, elevated B-cellpopulations, B-cell mediated autoimmune diseases, leukemias, andlymphomas (see e.g. Nitschke L, Glycobiology 24: 807-17 (2014)). Inaddition, CD22 might be overexpressed by a variety of malignant B-cells,such as, e.g., B-cell neoplasms for which the majority that have beenanalyzed expressed cell-surface CD22.

There are numerous CD22 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “CD22 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a CD22molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD22 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, CD2 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human CD22.

In certain embodiments, the CD22 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, CD22 binding region is derived from animmunoglobulin, CD22 binding region, such as an antibody paratopecapable of binding an extracellular part of CD22. In certain otherembodiments, the immunoglobulin-type, CD22 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, CD22 binding region by providinghigh-affinity binding to an extracellular part of CD22. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous CD22 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,CD22 binding regions include CD22-binding monoclonal antibodies andderivatives thereof, such as humanized variants and recombinantimmunoglobulin domains, e.g., RFB4, alpha S-HCL-1 (alpha Leu-14), HD39,To15, 4KB128, HD37, EPB, HD6, LL2, HA22-LR, 11B22.7, Hu10F4 (MCDT2219Aor pinatuzumab), epratuzumab, inotuzumab, CAT-3888 (BL22), CAT-8015(moxetumomab), and scFv-4KB128 (see e.g., Campana D et al., J Immunol134: 1524-30 (1985); Schwarting R et al., Blood 65: 974-83 (1985);Dorken B et al., J Immunol 136: 4470-9 (1986); Mason D et al., Blood 69:836-40 (1987); Ghetie M et al., Cancer Res 48: 2610-7 (1988);Pawlak-Byczkowska E et al., Cancer Res 49: 4568-77 (1989); Press O etal., Cancer Res 49: 4906-12 (1989); Stein R et al., Cancer ImmunolImmunother 37: 293-8 (1993); Leung S et al., Hybridoma 13: 469-76(1994); WO 1994/027638; Leung S et al., Mol Immunol 32: 1413-27 (1995);WO 1998/041641; WO 2000/074718; Coleman M et al., Clin Cancer Res 9:3991S-4S (2003); WO 2003/027135; WO 2003/072036; Arndt M et al., FEBSLett 578: 257-61 (2004); Furman et al., Curr Treat Options Oncol 5:283-8 (2004); WO 2005/012493; Ho M et al., Proc Natl Acad Sci USA 103:9637-42 (2006); U.S. Pat. No. 7,074,403; WO 2008/070569; O'Donnell etal., Caner Immunol Immunother 58: 1715-22 (2009); Mussai Et al., Br JHaematol 150: 352-8 (2010); Polson A et al., Leukemia 24: 1566-73(2010); Wayne et al., Clin Cancer Res 16: 1894-903 (2010); Wong et al.,Expert Opin Biol Ther 10: 1251-8 (2010); US 20140248278; JP201518404).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanCD22 and/or the cellular surface of a CD22+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HABR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:130, SEQ ID NO:136, SEQ ID NO:142, or SEQ IDNO:148; (ii) a HABR2 comprising or consisting essentially of one of theamino acid sequence as shown in SEQ ID NO:131, SEQ ID NO:137, SEQ IDNO:143, or SEQ ID NO:149; and (iii) a HABR3 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:132,SEQ ID NO:138, SEQ ID NO:144 or SEQ ID NO:150; and b) a light chainvariable (V_(L)) domain comprising (i) a LABR1 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:133,SEQ ID NO:139, SEQ ID NO:145, or SEQ ID NO:151; (ii) a LABR2 comprisingor consisting essentially of one of the amino acid sequence as shown inSEQ ID NO:134, SEQ ID NO:140, SEQ ID NO:146, or SEQ ID NO:152; and (iii)a LABR3 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:135, SEQ ID NO:141, SEQ ID NO:147, or SEQID NO:153. Alternatively, the binding regions could be described byCDRs, which largely overlap with ABRs and are described in SEQ ID NOs:154-165. In certain further embodiments, the binding region comprises orconsists essentially of amino acids 269-513 of SEQ ID NO: 40 or 80.

According to one specific but non-limiting aspect, the binding region ofthe cell-targeting molecule of the present invention comprises a ligand(whether naturally occurring or synsthetic) or derivative thereof thatretains binding functionality to CD22, such as, e.g., sialic acids,sialic acid-containing glycoconjugates, and domains of soluble type-Mimmunoglobulins (IgMs) (see e.g. Bakker T et al., Eur J Immunol 32:1924-32 (2002); Chen W et al., Blood 115: 4778-86 (2010); Chen W et al.,Leuk Lymphoma 53: 208-10 (2012); Schweizer A et al., Eur J Immunol 42:2792-802 (2012)). Synthetic CD22 ligands with high binding affinitieshave been designed and can be used for cell-targeting (see e.g., Razi N,Varki A et al., Proc Natl Acad Sci USA 95: 7469-74 (1998); Sliedregt Let al., Bioorg Med Chem 9: 85-97 (2001); van Rossenberg S et al., J BiolChem 276: 12967-73 (2001); Kelm S et al., J Exp Med 195: 1207-13 (2002);Collins B et al., J Immunol 177: 2994-3003 (2006); Yu J et al., BiochemBiophys Res Commun 360: 759-64 (2007); Abdu-Allah H et al., J Med Chem51: 6665-81 (2008); O'Reilly M et al., J Am Chem Soc 130: 7736-45(2008); Abdu-Allah H et al., Bioorg Med Chem Lett 19: 5573-5 (2009);Chen W et al., Blood 115: 4778-86 (2010); Lepenies B et al., Curr OpinChem Biol 14: 404-11 (2010); Abdu-Allah H et al., Bioorg Med Chem 19:1966-71 (2011); Chen W et al., Leuk Lymphoma 53: 208-10 (2012); Mesch Set al., ChemMedChem 7: 134-43 (2012); Kelm S et al., Angew Chem Int EdEngl 52: 3616-20 (2013); Macauley M et al., J Clin Invest 123: 3074-83(2013); Preshcer H et al., ACS Chem Biol 9: 1444-50 (2014)).

Any of the aforementioned CD22 binding molecules may be suitable for useas a CD22 binding region or modified to create one or more CD22 bindingregions for use in a cell-targeting molecule of the present invention.

4. Cell-Targeting Molecules Targeting Human CD30

CD30, also recognized in the art as tumor necrosis factor receptorsuperfamily 8 (TNFRSF8) or Ki-1/120, is a type I transmembraneglycoprotein of a size of about 90 to 120 kDa. CD30 functions as acell-surface receptor (or co-receptor) of the tumor necrosis factorreceptor family and binds a ligand, CD30L. A CD30 antigen was firstdescribed as a marker of classical Hodgkin lymphoma and Reed-Sternbergcells present in patients with Hodgkin's disease (Schwab U et al.,Nature 299: 65-7 (1982); Stein H et al., Int J Cancer 30: 445-459(1982)), and CD30 antigen was later observed on non-Hodgkin lymphomacells (see e.g. Stein H et al., Blood 66: 848-58 (1985)). While the nameCD30 might refer to multiple proteins with related structures andpolypeptide sequences from various species, for the purposes of thestructural examples of this section, the term “CD30” refers to the tumornecrosis factor receptor proteins present in humans whose exact sequencemight vary slightly based on the isoform and from individual toindividual. With regard to humans, CD30 refers to the proteinrepresented by the predominant polypeptide sequence UniProt P28908 andNCBI accession AAA51947.1; however, different isoforms and variants mayexist due to splicing, polymorphisms and/or mutations. A skilled workerwill be able to identify other CD30 proteins in humans, even if theydiffer from the referenced sequences.

CD30 is an attractive target for cell-targeted therapeutics, e.g.,because its expression is largely restricted to activated and/orproliferating lymphocytes and malignant cells. In normal or inflamedtissues, CD30 expression is largely restricted to medium/large activatedB-cells and/or activated T-cells which produce Th2-type cytokines(Chiarle R et al., Clin Immunol 90: 157-64 (1990); Werner B et al., JCutan Pathol 35: 1100-7 (2008); Buchan S, A1-Shamkhani A, PLoS One 7:e45244 (2012)). CD30 is highly expressed by certain cell types, such as,e.g., certain lymphoma cells, other malignant lymphoid cells, andnon-lymphoid tumor cells, whereas only a restricted subset of healthycells express CD30 and at lower levels (Deutsch Y et al., Leuk Lymphoma52: 1641-54 (2011)). CD30 is expressed by cells involved inlymphoproliferative disorders, lymphoid neoplasms, and myeloidneoplasms. For example, CD30 is expressed on a subset of non-Hodgkinlymphoma cells, including Burkitt's, anaplastic large-cell lymphomacells (ALCL), T-cell lymphomas, cutaneous T-cell lymphoma cells, nodularsmall cleaved-cell lymphoma cells, lymphocytic lymphoma cells,peripheral T-cell lymphoma cells, Lennert's lymphoma cells,immunoblastic lymphoma cells, T-cell leukemidymphoma cells (ATLL), adultT-cell leukemia (T-ALL), centroblastic/centrocytic (cb/cc) follicularlymphoma cells, and lymphomatoid papulosis cells (see e.g., Stein H etal., Blood 66: 848-58 (1985); Stein et al., Neoplastic Hematophathology,pg 675, (Baltimore, Williams & Wilkins, Knowles D, ed.) (1985); Stein Het al., Pathology of Cells Receptors and Tumor Markers, pg 121(Stuttgart, Gustav Fischer Verlag, Sefert G, Hubner K (eds) (1987);Suchi T et al., J Clin Pathol 40: 995 (1987); Eckert F et al., Am JDermatopathol 11: 345-52 (1989); Moller P et al., Am J Clin Pathol 91:18-23 (1989); Burns B, Dardick I, Am J Clin Pathol 93: 327-32 (1990);Piris M et al., Histopathology 17: 211-8 (1990); Miettinen M, ArchPathol Lab Med 116: 1197-201 (1992); Norduyn L et al., J Clin Pathol 47:33-7 (1994); Sabattini E et al., Haematologica 98: e8 1-2 (2013)). CD30expression has been observed in embryonal carcinomas, nonembryonalcarcinomas, malignant melanomas, mesenchymal tumors, and myeloid celllines and macrophages at late stages of differentiation (see e.g.,Andreesen R et al., Blood 63: 1299-1302 (1984); Schaadt M et al., IntRev Exp Pathol 27: 185-202 (1985); Stein H et al., Haematol BloodTransfus 29: 441-4 (1985); Froese P et al., J Immunol 139: 2081-7(1987); Pallesen G, Hamilton-Dutoit S, Am J Pathol 133: 446-50 (1988);Andreesen R et al., Am J Pathol 134: 187-92 (1989); Hansen H et al.,Biol Chem Hoppe-Seyler 370: 409-16 (1989); Schwarting R et al., Blood74: 1678-89 (1989); Mechtersheimer G, Möller P, Cancer 66: 1732-7(1990); Pallesen G, Histopathology 16: 409-13 (1990); Dürkop H et al.,Cell 68: 421-7 (1992); Latza U et al., Am J Pathol 146: 463-71 (1995)).CD30 expression appears upregulated by neoplastic mast cells of advancedneoplasms, such as, neoplasms involved in mastocytosis and systemicmastocytosis (see e.g., Soltar K et al., Mod Pathol 24: 585-95 (2011);Valent P et al., Leuk Lymphoma 52: 740-4 (2011)). CD30 expression alsohas been reported to increase in a variety of autoimmune andinflammatory diseases, such as, e.g., lymphoid neoplasms, myeloidneoplasms, atopic allergies (atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis), systemic lupus erythematosus,systemic sclerosis (scleroderma), graft-versus-host disease, HIVinfection, Epstein-Barr virus infection, measles, mononucleosisinfection, Omen's syndrome, ulcerative colitis, rheumatoid arthritis,multiple sclerosis, psoriasis, Hashimoto's thyroiditis, primary biliarycirrhosis, Sjögren's syndrome, toxoplasmosis, Wegener's granulomatosis,and tuberculosis (see e.g., Ralfkiaer E et al., Arch Dermatol Res 279:28-292 (1987); Romagnani S et al., J Leukocyte Biol 57: 726-30 (1995);Gruss H et al., Immunol Today 18: 156-63 (1997); Horie R, Watababe T,Semin Immunol 10: 457-70 (1998); Bengtsson A, Allergy 561: 593-603(2001); Gerli R et al., Trends Immunol 22: 72-7 (2001)). CD30 expressionis a marker for mastocytosis (see e.g. Maric J, Calvo K, Leuk Lymphoma52: 732-3 (2011)).

There are numerous CD30 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “CD30 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a CD30molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD30 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, CD30 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human CD30.

In certain embodiments, the CD30 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, CD30 binding region is derived from animmunoglobulin, CD30 binding region, such as an antibody paratopecapable of binding an extracellular part of CD30. In certain otherembodiments, the immunoglobulin-type, CD30 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, CD30 binding region by providinghigh-affinity binding to an extracellular part of CD30. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous CD30 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,CD30 binding regions include CD30-binding monoclonal antibodies andderivatives thereof, such as humanized variants and recombinantimmunoglobulin domains, e.g., Ki-1, HeFi-1, Ber-H2, Ber-H4, Ber-H6,Ber-H8, Ber-H10, HRS-1, HRS-3, HRS-4, AC10, C10, Ki-2, Ki-3, Ki-4, Ki-5,Ki-6, Ki-7, M44, M67, scFv-Ki-4, scFv 4E3, T6, T7, T13, T14, T21, T24,T25, T104, T105, T107, T112, T201, T214, T215, T405, T406, T408, T411,T420, T426, T427 (see e.g., Schwab U et al., Nature 299: 65-7 (1982);Hecht T et al., J Immunol 134: 4231-6 (1985); Schwarting R et al., IssueSections. In: J. A. McMichael (ed.). Leucocyte Typing 3: 574-75. Oxford:Oxford University Press, (1987); Schwarting R et al., Leucocyte TypingIV: 419-22. Oxford, UK, Oxford University (1989); Bowen M et al., JImmunol 151: 5896-906 (1993); Gruss H et al., Blood 83: 2045-56 (1994);Horn-Lohrens O et al., Int J Cancer 60: 539-44 (1995); WO 1996/022384;Barth S et al., Blood 95: 3909-14 (2000); Klimka A et al., Br J Cancer83: 252-60 (2000); WO 2002/043661; WO 2003/059282; US 2004/018194; WO2005/001038; WO 2007/040653; WO 2008/025020; WO 2015/028444).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanCD30 and/or the cellular surface of a CD30+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HABR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:166, SEQ ID NO:172, SEQ ID NO:178, or SEQ IDNO:184; (ii) a HABR2 comprising or consisting essentially of one of theamino acid sequence as shown in SEQ ID NO:167, SEQ ID NO:173, SEQ IDNO:179, or SEQ ID NO:185; and (iii) a HABR3 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:168,SEQ ID NO:174, or SEQ ID NO:180; and b) a light chain variable (V_(L))domain comprising (i) a LABR1 comprising or consisting essentially ofone of the amino acid sequence as shown in SEQ ID NO:169, SEQ ID NO:175,SEQ ID NO:181 or SEQ ID NO:186; (ii) a LABR2 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:170,SEQ ID NO:176, SEQ ID NO: 182, or SEQ ID NO:187; and (iii) a LABR3comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:171, SEQ ID NO:177, SEQ ID NO:183, or SEQ IDNO:188. In certain further embodiments, the cell-targeting molecule ofthe present invention comprises the binding region comprising orconsisting essentially of amino acids 268-500 of any one of SEQ ID NOs:452, 472, 487, and 503.

According to one specific but non-limiting aspect, the binding region ofthe cell-targeting molecule of the present invention comprises a ligand(whether naturally occurring or synthetic) or a derivative thereof thatretains binding functionality to an extracellular part of CD30 (see e.g.Powell I et al., J Leukoc Biol 63: 752-7 (1998); Gruss H et al., Eur JImmunol 25: 2083 (1995); Gattei V et al., Leuk Lymphoma 35: 21-35(1999); Zhang P et al., Lab Invest 89: 1423-32 (2009); Parekh P et al.,Biomaterials 34: 8909-17 (2013); Shinoda K et al., J Autoimmun 57: 14-23(2015); WO 1993/024135).

Any of the aforementioned CD30 binding molecules may be suitable for useas a CD30 binding region or modified to create one or more CD30 bindingregions for use in a cell-targeting molecule of the present invention.

5. Cell-Targeting Molecules Targeting Human CD38

CD38 is transmembrane protein characterized as both a cell surfacereceptor and extracellular cyclic ADP ribose hydrolase (ADP-ribosylase).While the name CD38 might refer to multiple proteins with relatedstructures and polypeptide sequences from various species, for thepurposes of the structural examples of this section, the term “CD38”refers to the cyclic ADP ribose hydrolase proteins present in humanswhose exact sequence might vary slightly based on the isoform and fromindividual to individual. With regard to humans CD38 refers to theprotein represented by the predominant polypeptide sequence UniProtP28907 and NCBI accession BAA18964; however, different isoforms andvariants may exist due to splicing, polymorphisms and/or mutations (seee.g. Ferrero E et al., Immunogenetics 49: 597-604 (1999);Gonzalez-Escribano M et al., Hum Immunol 65: 660-664 (2004); Drummond Fet al., J Bone Miner Metab 24: 28-35 (2006); Aydin S et al., Blood 111:5646-53 (2008); WO 2006/099875). A skilled worker will be able toidentify other CD38 proteins in humans, even if they differ from thereferenced sequences.

There are numerous CD38 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “CD38 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a CD38molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD38 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, CD38 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human CD38.

In certain embodiments, the CD38 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, CD38 binding region is derived from animmunoglobulin, CD38 binding region, such as an antibody paratopecapable of binding an extracellular part of CD38. In certain otherembodiments, the immunoglobulin-type, CD38 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, CD38 binding region by providinghigh-affinity binding to an extracellular part of CD38. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous CD38 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,CD38 binding regions include CD38-binding monoclonal antibodies andscFvs such as, e.g daratumumab, isatuximab, and MOR202 (see e.g. DeaglioS et al., Trends Mol Med 14: 210-8 (2008); van de Donk N et al., ImmunolRev 270: 95-112 (2016); WO 1996/016990; WO 2002/006347; WO 2005/103083;WO 2008/047242; WO 2012/092612; WO 2012/092616; US20020164788;US20100285004; US 20150118251).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanCD38 and/or the cellular surface of a CD38+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HABR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:189, SEQ ID NO:195, SEQ ID NO:201, SEQ ID NO:207,SEQ ID NO:213, or SEQ ID NO:219; (ii) a HABR2 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:190,SEQ ID NO:196, SEQ ID NO:202, SEQ ID NO:208, SEQ ID NO:214, or SEQ IDNO:220; and (iii) a HABR3 comprising or consisting essentially of one ofthe amino acid sequence as shown in SEQ ID NO:191, SEQ ID NO:197, SEQ IDNO:203, SEQ ID NO:209, SEQ ID NO:215, or SEQ ID NO:221; and b) a lightchain variable (V_(L)) domain comprising (i) a LABR1 comprising orconsisting essentially of one of the amino acid sequence as shown in SEQID NO:192, SEQ ID NO:198, SEQ ID NO:204, SEQ ID NO:210, SEQ ID NO:216,or SEQ ID NO:222; (ii) a LABR2 comprising or consisting essentially ofone of the amino acid sequence as shown in SEQ ID NO:193, SEQ ID NO:199,SEQ ID NO:205, SEQ ID NO:211, SEQ ID NO:217, or SEQ ID NO:223; and (iii)a LABR3 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:194, SEQ ID NO:200, SEQ ID NO:206, SEQ IDNO:212, SEQ ID NO:218, or SEQ ID NO:224. Alternatively, the bindingregions could be described by CDRs, which largely overlap with ABRs andare described in SEQ ID NOs: 225-242. In certain further embodiments,the binding region comprises or consists essentially of amino acids269-499, 269-512, 269-513, or 280-510 of any one of SEQ ID NOs: 34, 35,41-56, and 82.

A natural CD38 ligand or derivative thereof may be utilized as thebinding region of a cell-targeting molecule of the present invention.Native CD38 is known to bind at least one ligand, CD38L, an Ig proteinalso known as platelet endothelial cell adhesion molecule 1 (PECAM1) orCD31 (Cesano A et al., J Immunol 160: 1106-15 (1998); Deaglio S et al.,J Immunol 160: 395-402 (1998)). CD31 or a part of CD31 that interactswith CD38 or a derivative thereof may be fused to Shiga toxin effectorpolypeptides of the invention to construct CD38-targeting,cell-targeting molecules that bind an extracellular part of CD38.

Any of the aforementioned CD38 binding molecules may be suitable for useas a CD38 binding region or modified to create one or more CD38 bindingregions for use in a cell-targeting molecule of the present invention.

6. Cell-Targeting Molecules Targeting Human CD45

CD45, also recognized in the art as PTPRC (protein tyrosine phosphatase,receptor type, C) and leukocyte common antigen (LCA), is a type Itransmembrane, protein tyrosine phosphatase expressed on the cellsurfaces of many differentiated hematopoietic cells, especiallymalignant, hematological cells, such as, e.g., lymphoma, B-cell chroniclymphocytic leukemia (B-CLL), hairy cell leukemia, and acutenonlymphocytic leukemia (AML) cells. While the name CD45 might refer tomultiple proteins with related structures and polypeptide sequences fromvarious species, for the purposes of the structural examples of thissection, the term “CD45” refers to the protein tyrosine phosphataseproteins present in humans whose exact sequence might vary slightlybased on the isoform and from individual to individual. With regard tohumans, CD45 refers to the protein represented by the predominantpolypeptide sequence UniProt Q6QIQ5 and, e.g., NCBI accessionNP_563578.2 or NP_002829.3; however, different isoforms and variantsexist due to splicing, polymorphisms and/or mutations (see e.g.Motta-Mena L et al., J Biol Chem 286: 20043-53 (2011); Marme F et al.,Breast Cancer Res Treat 132: 819-31 (2012); Pokoyski C et al., GenesImmun 16: 519-27 (2015)). A skilled worker will be able to identifyother CD45 proteins in humans, even if they differ from the referencedsequences.

There are numerous CD45 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “CD45 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a CD45molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD45 of 10⁻⁵ to 10⁻¹² moles per liter. As usedherein, CD45 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human CD45.

In certain embodiments, the CD45 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, CD45 binding region is derived from animmunoglobulin, CD45 binding region, such as an antibody paratopecapable of binding an extracellular part of CD45. In certain otherembodiments, the immunoglobulin-type, CD45 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, CD45 binding region by providinghigh-affinity binding to an extracellular part of CD45. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous CD45 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,CD45 binding regions include CD45-binding monoclonal antibodies andscFvs, such as, e.g., anti-CD45RB (see e.g., Luke P et al., Curr Mol Med1: 533-43 (2001); Lin Y et al., Cancer Res 66: 3884-92 (2006)).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanCD45 and/or the cellular surface of a CD45+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HABR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:243, SEQ ID NO:249, or SEQ ID NO:255; (ii) a HABR2comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:244, SEQ ID NO:250, or SEQ ID NO:256; and (iii) aHABR3 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:245, SEQ ID NO:251, or SEQ ID NO:257; andb) a light chain variable (V_(L)) domain comprising (i) a LABR1comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:246, SEQ ID NO:252, or SEQ ID NO:258; (ii) a LABR2comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:247, SEQ ID NO:253, or SEQ ID NO:259; and (iii) aLABR3 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:248, SEQ ID NO:254, or SEQ ID NO:260.

Any of the aforementioned CD45 binding molecules may be suitable for useas a CD45 binding region or modified to create one or more CD45 bindingregions for use in a cell-targeting molecule of the present invention.

7. Cell-Targeting Molecules Targeting Human HER2

HER2, also recognized in the art as Receptor tyrosine-protein kinaseerbB-2, is a transmembrane protein which functions as a cell surfacereceptor for transducing signals across the cellular membrane tointracellular regulators of cell proliferation and apoptosis. HER2 isalso recognized in the art as Neu, erbB-2, p185, CD340, NGL, andHER2/neu (Coussens L et al., Science 230: 1132-39 (1985); King C et al.,Science 229: 974-6 (1985); Semba K et al., Proc Natl Acad Sci USA 82:6497-501 (1985); Yamamoto T et al., Nature 319:230-234 (1986); Kokai Yet al., Proc Natl Acad Sci USA 85: 5389-93 (1988); Disis M et al, CancerRes 54: 16-20 (1994); Yoshino I et al., J Immunol 152: 2393-400 (1994)see, e.g., GenBank Acc. Nos. X03363; M17730; NM_004448; SEG_HLMHER20).While the name HER2 might refer to multiple proteins with relatedstructures and polypeptide sequences from various species, for thepurposes of the structural examples of this section, the term “HER2”refers to the epidermal growth factor receptor proteins present inhumans whose exact sequence might vary slightly based on the isoform andfrom individual to individual. For example, HER2 refers to the humanprotein represented by the exemplary polypeptide sequences UniProtP04626 and NCBI accessions NP_004439.2, NP_001005862.1, NP_001276865.1,NP_001276866.1, and NP_001276867.1; however, different isoforms andvariants exist due to splicing, polymorphisms and/or mutations (see e.g.Siddig A et al., Ann NY Acad Sci 1138: 84-94 (2008); Poole E et al., IntJ Mol Epidemiol Genet 2: 300-15 (2011); WO 2000/020579). A skilledworker will be able to identify other HER2 proteins in humans, even ifthey differ from the referenced sequences.

HER2 is overexpressed by many cancer cells, notably breast cancer cells,and its overexpression is strongly associated with increased metastasis,increased disease reoccurrence, and poor prognosis (see e.g. Slamon D etal., Science 235: 177-82 (1987)).

There are numerous HER2 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “HER2 bindingregion” refers to a molecular moiety (e.g. a proteinaceous molecule) oragent capable of specifically binding an extracellular part of a HER2molecule with high affinity, such as, e.g., having a dissociationconstant with regard to CD20 of 10⁻⁵ to 10−12 moles per liter. As usedherein, HER2 binding refers to the ability to bind to an extracellularpart of an isoform or variant of human HER2.

In certain embodiments, the HER2 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, HER2 binding region is derived from animmunoglobulin, HER2 binding region, such as an antibody paratopecapable of binding an extracellular part of HER2. In certain otherembodiments, the immunoglobulin-type, HER2 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, HER2 binding region by providinghigh-affinity binding to an extracellular part of HER2. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions and/orantigen binding regions from immunoglobulins as described herein.

There are numerous HER2 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,HER2 binding regions include HER2-binding monoclonal antibodies andderivatives thereof, such as, e.g., anti-ErbB2, 4D5, 2C4, 7F3, 7C2,mumAb 4D5, chmAb 4D5, (rhu)mAb 4D5, huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, huMAb4D5-8, trastuzumab,humanized 520C9, 4D5Fc8, hingeless rhu4D5, non-glycosylated rhu4D5 withmutated cysteine residues, pertuzumab, and humanized 2C4 (Hudziak R etal., Mol Cell Biol 9: 1165-72 (1989); McKenzie S et al., Oncogene4:543-8 (1989); Bacus S et al., Molecular Carcinogenesis 3: 350-62(1990); Hancock M et al., Cancer Res 51: 4575-80 (1991); Maier L et al.,Cancer Res 51: 5361-5369 (1991); Stancovski I et al., Proc Natl Acad SciUSA 88: 8691-5 (1991); Tagliabue E et al., Int J Cancer 47: 933-937(1991); Bacus S et al., Cancer Res 52: 2580-9 (1992); Carter P et al.,Proc Natl Acad Sci USA 89: 4285-89 (1992); Harwerth I et al. J Biol Chem267: 15160-7 (1992); Kasprzyk P et al., Cancer Res 52: 2771-6 (1992);Lewis G et al., Cancer Immunol Immunother 37: 255-63 (1993); Xu F etal., Int J Cancer 53: 401-8 (1993); Arteaga C et al., Cancer Res 54:3758-65 (1994); Shawyer L et al., Cancer Res 54: 1367-73 (1994); KlapperL et al. Oncogene 14: 2099-109 (1997); WO 1993/21319; WO 1994/00136; WO1997/00271; WO 1998/77797; U.S. Pat. Nos. 5,772,997; 5,783,186;5,821,337; 5,840,525; 6,949,245; and 7,625,859).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanHER2 and/or the cellular surface of a HER2+ cell. In certain embodimentsof the cell-targeting molecule of the present invention, the bindingregion comprises a polypeptide(s) selected from the group consisting of:a) a heavy chain variable (V_(H)) domain comprising (i) a HABR1comprising or consisting essentially of one of the amino acid sequencesas shown in SEQ ID NO:261, SEQ ID NO:268, or SEQ ID NO:274; (ii) a HABR2comprising or consisting essentially of one of the amino acid sequenceas shown in SEQ ID NO:262, SEQ ID NO:269, or SEQ ID NO:275; and (iii) aHABR3 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:270, or SEQID NO:276; and b) a light chain variable (V_(L)) domain comprising (i) aLABR1 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:264, SEQ ID NO:271, or SEQ ID NO:277;(ii) a LABR2 comprising or consisting essentially of one of the aminoacid sequence as shown in SEQ ID NO:265, SEQ ID NO:272, or SEQ IDNO:278; and (iii) a LABR3 comprising or consisting essentially of one ofthe amino acid sequence as shown in SEQ ID NO:266, SEQ ID NO:273, or SEQID NO:279. Alternatively, the binding regions could be described byCDRs, which largely overlap with ABRs and are described in SEQ ID NOs:283-303. In other embodiments of the cell-targeting molecule of thepresent invention, the binding region comprises a polypeptide(s)selected from the group consisting of: a) a heavy chain only variable(V_(H)H) domain comprising (i) a HABR1 comprising or consistingessentially of the amino acid sequences as shown in SEQ ID NO:280; (ii)a HABR2 comprising or consisting essentially of the amino acid sequenceas shown in SEQ ID NO:281; and (iii) a HABR3 comprising or consistingessentially of the amino acid sequence as shown in SEQ ID NO:282. Incertain further embodiments, the binding region comprises or consistsessentially of amino acids 269-520 or 269-521 of any one of SEQ ID NOs:36, 66, and 67.

A natural ligand or derivative thereof may be utilized as the HER2binding region for a cell-targeting molecule of the present invention.Native HER2 is known to heterodimerize with other members of the ErbBfamily upon binding ligands such as epidermal growth factors likeepiregulin and heregulin (Moasser M, Oncogene 26: 6469-87 (2007); RieseD, Cullum R, Semin Cell Dev Biol 28: 49-56 (2014); Sollome J et al.,Cell Signal 26: 70-82 (2014)). ErbB ligands which bind members of theErbB family include EGF, TGF-α, amphiregulin, betacellulin, HB-EGF,epiregulin, HER2-68 and HER2-100, heregulins, herstatin, NRG-2, NRG-3,and NRG-4 (Justman Q et al., J Biol Chem 277: 20618-24 (2002);Jhabvala-Romero F., et al., Oncogene 22: 8178-86 (2003)). Examples of anErbB ligand include the heregulins (HRG), such as the prototypeheregulin disclosed in U.S. Pat. No. 5,641,869 and Marchionni M et al.,Nature 362: 312-8 (1993). Examples of heregulins include heregulin-α,heregulin-β1, heregulin-β2 and heregulin-β3 (Holmes W et al., Science256: 1205-10 (1992); U.S. Pat. No. 5,641,869); neu differentiationfactor (NDF) (Peles et al., Cell 69: 205-16 (1992)); acetylcholinereceptor-inducing activity (ARIA) (Falls D et al., Cell 72: 801-15(1993)); glial growth factors (GGFs) (Marchionni M et al., Nature 362:312-8 (1993)); sensory and motor neuron derived factor (SMDF) (Ho W etal., J Biol Chem 270: 14523-32 (1995)); γ-heregulin (Schaefer G et al.,Oncogene 15: 1385-94 (1997)).

An ErbB ligand according to the present invention may also be asynthetic ErbB ligand. The synthetic ligand may be specific for aparticular ErbB receptor, or may recognize particular ErbB receptorcomplexes. An example of a synthetic ligand is the syntheticheregulin/EGF chimera biregulin (Jones J et al., FEBS Lett, 447: 227-31(1999)) and the EGF-like domain fragment HRGβ1177-244. ErbB ligands or apart of an ErbB ligand that interacts with HER2 or a derivative thereofmay be fused to Shiga toxin effector polypeptides of the invention toconstruct HER2-targeting, cell-targeting molecules of the invention thatbind an extracellular part of HER2.

Synthetic peptides which bind an extracellular part of HER2 may beutilized as the binding region for targeting. Many peptides have beendescribed which are capable of binding to HER2 (see e.g. U.S. Pat. Nos.5,578,482; 5,856,110; 5,869,445; 5,985,553; 6,333,169; 6,987,088;7,019,017; 7,282,365; 7,306,801; 7,435,797; 7,446,185; 7,449,480;7,560,111; 7,674,460; 7,815,906, 7,879,325; 7,884,194; 7,993,650;8,241,630; 8,349,585; 8,389,227; 8,501,909; 8,512,967; 8,652,474; and US2011/0059090).

In certain embodiments, small molecules which bind an extracellular partof HER2 may be utilized as the binding region for targeting. Many smallmolecules have been described which are capable of binding to HER2 suchas tyrosine kinase inhibitors, AZD8931, lapatinib, neratinib (HKI-272),dacomitinib (PF-00299804), afatinib (BIBW 2992) (Barlaam B et al., ACSMed Chem Lett 4: 742-6 (2013); Yu H, Riley G, J Natl Compr Canc Netw 11:161-9 (2013); Roskoski R, Pharmacol Res 87C: 42-59 (2014)). Other smallmolecules which bind to an extracellular part of HER2 may be identifiedusing methods well known to those of skill in the art, such as byderivatizing known EGFR binders like gefitinib, erlotinib, AEE788,AG1478, AG1571 (SU-5271), AP26113, CO-1686, XL647, vandetanib, andBMS-690514 (Kurokawa H, Arteaga C, Clin Cancer Res 7: 4436s-4442s(2001); Yigitbasi O et al., Cancer Res 64: 7977-84 (2004); Yu H, RileyG, J Natl Compr Canc Netw 11: 161-9 (2013); Roskoski R, Pharmacol Res87C: 42-59 (2014)).

Any of the aforementioned HER2 binding molecules may be suitable for useas a HER2 binding region or modified to create one or more HER2 bindingregions for use in a cell-targeting molecule of the present invention

8. Cell-Targeting Molecules Targeting Human PD-L1

PD-L1, also recognized in the art as PDL1, programmed cell death 1ligand 1, PDCD1 Ligand 1, PDCD1L1, PDCD1LG1, B7 homolog 1 (B7-H1), andCD274, is a ligand for the programmed cell death-1 receptor of T- andB-cells (Dong H et al., Nat Med 5: 1365-9 (1999); Freeman G et al., JExp Med 192: 1027-34 (2000); Latchman Y et al., Nat Immunol 2: 261-8(2001)) and the B7-1 receptor and CD80 receptor found on T-cells (ButteM et al., Immunity 27:111-22 (2007); Park J et al., Blood 116: 1291-8(2010)). While the name PD-L1 might refer to multiple proteins withrelated structures and polypeptide sequences from various species, forthe purposes of the structural examples of this section, the term“PD-L1” refers to the PD-1 ligands present in humans whose exactsequence might vary slightly based on the isoform and from individual toindividual. With regard to humans, PD-L1 refers to the proteinrepresented by the predominant polypeptide sequence UniProt Q9NZQ7 orQ9EP73 and NCBI accession AAI13735.1; however, different isoforms andvariants exist due to splicing, polymorphisms and/or mutations (see e.g.Abelson A et al., Genes Immun 8: 69-74 (2007); Wang S et al., J ClinImmunol 27: 563-7 (2007); Hayashi M et al., Eur J Endocrinol 158: 817-22(2008); Mitchell A et al., J Clin Endocrinol Metab 94: 5139-45 (2009);Yang Q et al., Clin Exp Rheumatol 29: 13-8 (2011); Ma Y et al., Int JClin Exp Med 15: 16585-91 (2015)). A skilled worker will be able toidentify other PD-L1 proteins in humans, even if they differ from thereferenced sequences.

PD-L1 is absent from most healthy tissues under normal conditions;however, PD-L1 expression can be induced by exposure of most nucleatedmammalian cells to an interferon(s) (see e.g., Dong H et al., Nat Med 8:793-800 (2002); Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F etal., Cancer Res 65: 1089-96 (2005); Zou W, Chen L, Nat Rev Immunol 8:467-7 (2008); Flies D et al., Yale J Biol Med 84: 409-21 (2011); Chen Jet al., Immunobiology 217: 385-93 (2012); Spranger S et al. Sci TranslMed 5: 200ra116 (2013)). During certain malignancies, PD-L1 upregulationin tumor microenvironments might lead to over-suppression of immuneresponses to tumor cells, which fits common notions about PD-L1'sinvolvement in adaptive immune resistance of tumor cells to a host'simmune system (see e.g., Zou W, Chen L, Nat Rev Immunol 8: 467-7 (2008);Zheng P, Zho Z, Biomark Cancer 7: 15-8 (2015)).

PD-L1 is an attractive target for therapies because PD-L1 is stronglyexpressed by certain tumor cells and tumor-infiltrating lymphocyteswhereas healthy, human tissues and cells seldom express high levels ofPD-L1 at a cellular surface (see e.g. Dong H et al., Nat Med 8: 793-800(2002); Chen L, Nat Rev Immunol 4: 336-47 (2004); Hirano F et al.,Cancer Res 65: 1089-96 (2005); Chen L, Han X, J Clin Invest 125: 3384-91(2015)). In humans, expression of cell-surface PD-L1 by tumor cells hasbeen observed in a number of primary tumor biopsies and tumor-cellcultured cells assessed by immunohistochemistry, including cells andtissues related to, e.g., carcinomas, gliomas, B-cell lymphomas, adultT-cell leukemia/lymphoma (ATLL), angioimmunoblastic T-cell lymphomas(AITLs), bladder cancers, chronic lymphocytic leukemias (CLLs),epithelial malignancies, oral squamous cell carcinomas, esophagealsquamous cell carcinomas (ESCCs), lung cancers, non-Hodgkin lymphomas(NHLs), pancreatic cancers, renal cell carcinomas (RCCs), smalllymphocytic lymphomas (SLLs), squamous cell carcinomas of the head andneck (SCCHN), and virus-associated malignancies (see e.g., Brown J etal., Immunol 170: 1257-66 (2003); Strome S et al., Cancer Res 63: 6501-5(2003); Wintterle et al., Cancer Res 63: 7462-7 (2003); Thompson R etal., Cancer Res 66: 3381-5 (2006); Nomi T et al., Clin Cancer Res 13:2151-7 (2007); Thompson et al., Clin Cancer Res 13: 1757-61 (2007);Andorsky D et al., Clin Cancer Res 17: 4232-44 (2011); Chen B et al.,Clin Cancer Res 19: 3462-73 (2013); Chen M et al., Oncotarget 7: 7913-24(2016); Wu C et al., Sci Rep 6: 19740 (2016)).

There are numerous PD-L1 binding regions known to the skilled workerwhich may be associated with a Shiga toxin effector polypeptide of thepresent invention to create a cell-targeting molecule of the presentinvention. For purposes of the present invention, the term “PD-L1binding region” refers to a molecular moiety (e.g. a proteinaceousmolecule) or agent capable of specifically binding an extracellular partof a PD-L1 molecule with high affinity, such as, e.g., having adissociation constant with regard to PD-L1 of 10⁻⁵ to 10⁻¹² moles perliter. As used herein, PD-L1 binding refers to the ability to bind to anextracellular part of an isoform or variant of human PD-L1.

In certain embodiments, the PD-L1 binding region is animmunoglobulin-type binding region. In certain embodiments, theimmunoglobulin-type, PD-L1 binding region is derived from animmunoglobulin, PD-L1 binding region, such as an antibody paratopecapable of binding an extracellular part of PD-L1. In certain otherembodiments, the immunoglobulin-type, PD-L1 binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butwhich functions like an immunoglobulin, PD-L1 binding region byproviding high-affinity binding to an extracellular part of PD-L1. Thisengineered polypeptide may optionally include polypeptide scaffoldscomprising or consisting essentially of complementary determiningregions and/or antigen binding regions from immunoglobulins as describedherein.

There are numerous PD-L1 binding regions contemplated as components ofthe present invention. Non-limiting examples of immunoglobulin-type,PD-L1 binding regions include numerous antibodies and immunoglobulindomains already known to bind an extracellular part of PD-L1 present ata cellular surface include MDX-1105, MOM-18534-S(P), and various scFvs(see e.g., Latchman Y et al., Nat Immunol 2: 261-8 (2001); Xerri L etal., Human Pathol 39: 1050-8 (2008); Chen B et al., Clin Cancer Res 19:3462-73 (2013); Drees J et al., Protein Expr Purif 94: 60-6 (2014); WO2007/005874; WO 2010/036959; WO 2013/019906; U.S. Pat. Nos. 7,943,743;8,552,154; 9,102,727; 9,273,135, and U.S. patent applicationsUS20110271358 and US20160075782).

In certain embodiments, the cell-targeting molecule of the presentinvention comprises a binding region comprising an immunoglobulin-typepolypeptide selected for specific and high-affinity binding to humanPD-L1 and/or the cellular surface of a PD-L1+ cell. In certainembodiments of the cell-targeting molecule of the present invention, thebinding region comprises a polypeptide(s) selected from the groupconsisting of: a) a heavy chain variable (V_(H)) domain comprising (i) aHCDR1 comprising or consisting essentially of one of the amino acidsequences as shown in SEQ ID NO:304, SEQ ID NO:310, SEQ ID NO:316, SEQID NO:322, or SEQ ID NO:328; (ii) a HCDR2 comprising or consistingessentially of one of the amino acid sequence as shown in S SEQ IDNO:305, SEQ ID NO:311, SEQ ID NO:317, SEQ ID NO:323, or SEQ ID NO:329;and (iii) a HCDR3 comprising or consisting essentially of one of theamino acid sequence as shown in SEQ ID NO:306, SEQ ID NO:312, SEQ IDNO:318, SEQ ID NO:324, or SEQ ID NO:330; and b) a light chain variable(V_(L)) domain comprising (i) a LCDR1 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:307,SEQ ID NO:313, SEQ ID NO:319, SEQ ID NO:325, or SEQ ID NO:331; (ii) aLCDR2 comprising or consisting essentially of one of the amino acidsequence as shown in SEQ ID NO:308, SEQ ID NO:314, SEQ ID NO:320, SEQ IDNO:326, or SEQ ID NO:332; and (iii) a LCDR3 comprising or consistingessentially of one of the amino acid sequence as shown in SEQ ID NO:309,SEQ ID NO:315, SEQ ID NO:321, SEQ ID NO:327, or SEQ ID NO:333. Incertain further embodiments, the binding region comprises or consistsessentially of amino acids 269-498 or 269-499 of any one of SEQ ID NOs:37-39, 68-79, and 81.

Any of the aforementioned PD-L1 binding molecules may be suitable foruse as a PD-L1 binding region or modified to create one or more PD-L1binding regions for use in a cell-targeting molecule of the presentinvention.

Other Structural Variations

In certain embodiments of the cell-targeting molecules of the presentinvention, the binding region has been previously described, e.g., in WO2005/092917, WO 2007/033497, US2009/0156417, JP4339511, EP1727827,DE602004027168, EP1945660, JP4934761, EP2228383, US2013/0196928, WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, US20150259428, 62/168,758,62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, andPCT/US2016/016580.

In certain further embodiments of the cell-targeting molecule of thepresent invention, the binding region comprises a polypeptide(s)selected from the group consisting of: (a) a heavy chain variable(V_(H)) domain comprising a HCDR1 comprising or consisting essentiallyof the amino acid sequences as shown in SEQ ID NO:334, a HCDR2comprising or consisting essentially of the amino acid sequences asshown in HCDR2, and a HCDR3 comprising or consisting essentially of theamino acid sequences as shown in SEQ ID NO:336; and (b) a light chainvariable (V_(L)) domain comprising a LCDR1 comprising or consistingessentially of the amino acid sequences as shown in SEQ ID NO:337, aLCDR2 comprising or consisting essentially of the amino acid sequencesas shown in SEQ ID NO:338, and a LCDR3 comprising or consistingessentially of the amino acid sequences as shown in SEQ ID NO:339.

It is within the scope of the present invention to use fragments,variants, and/or derivatives of the cell-targeting molecules of thepresent invention which contain a functional binding site to anyextracellular part of a target biomolecule, and even more preferablycapable of binding a target biomolecule with high affinity (e.g. asshown by K_(D)). For example, any binding region which binds anextracellular part of a target biomolecule with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter, preferably less than 200 nM, maybe substituted for use in making cell-targeting molecules of theinvention and methods of the invention.

The skilled worker will recognize that variations may be made to theShiga toxin effector polypeptides and cell-targeting molecules of thepresent invention, and polynucleotides encoding any of the former,without diminishing their biological activities, e.g., by maintainingthe overall structure and function of the Shiga toxin effectorpolypeptide, such as in conjunction with one or more 1) endogenousepitope disruptions which reduce antigenic and/or immunogenic potential,2) furin-cleavage motif disruptions which reduce proteolytic cleavage,and/or 3) embedded or inserted epitopes which reduce antigenic and/orimmunogenic potential or are capable of being delivered to a MHC Imolecule for presentation on a cell surface. For example, somemodifications may facilitate expression, facilitate purification,improve pharmacokinetic properties, and/or improve immunogenicity. Suchmodifications are well known to the skilled worker and include, forexample, a methionine added at the amino-terminus to provide aninitiation site, additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons, andbiochemical affinity tags fused to either terminus to provide forconvenient detection and/or purification. A common modification toimprove the immunogenicity of a polypeptide produced using anon-chordate system (e.g. a prokaryotic cell) is to remove, after theproduction of the polypeptide, the starting methionine residue, whichmay be formylated during production, such as, e.g., in a bacterial hostsystem, because, e.g., the presence of N-formylmethionine (fMet) mightinduce undesirable immune responses in chordates.

Also contemplated herein is the inclusion of additional amino acidresidues at the amino and/or carboxy termini of a Shiga toxin effectorpolypeptide of the present invention, a cell-targeting molecule of thepresent invention, or a proteinaceous component of a cell-targetingmolecules of the present invention, such as sequences for epitope tagsor other moieties. The additional amino acid residues may be used forvarious purposes including, e.g., facilitating cloning, facilitatingexpression, post-translational modification, facilitating synthesis,purification, facilitating detection, and administration. Non-limitingexamples of epitope tags and moieties are chitin binding proteindomains, enteropeptidase cleavage sites, Factor Xa cleavage sites, FIAsHtags, FLAG tags, green fluorescent proteins (GFP),glutathione-S-transferase moieties, HA tags, maltose binding proteindomains, myc tags, polyhistidine tags, ReAsH tags, strep-tags, strep-tagII, TEV protease sites, thioredoxin domains, thrombin cleavage site, andV5 epitope tags.

In certain of the above embodiments, the polypeptide sequence of theShiga toxin effector polypeptides and/or cell-targeting molecules of thepresent invention are varied by one or more conservative amino acidsubstitutions introduced into the polypeptide region(s) as long as allrequired structural features are still present and the Shiga toxineffector polypeptide is capable of exhibiting any required function(s),either alone or as a component of a cell-targeting molecule. As usedherein, the term “conservative substitution” denotes that one or moreamino acids are replaced by another, biologically similar amino acidresidue. Examples include substitution of amino acid residues withsimilar characteristics, e.g. small amino acids, acidic amino acids,polar amino acids, basic amino acids, hydrophobic amino acids andaromatic amino acids (see, for example, Table C). An example of aconservative substitution with a residue normally not found inendogenous, mammalian peptides and proteins is the conservativesubstitution of an arginine or lysine residue with, for example,ornithine, canavanine, aminoethylcysteine, or another basic amino acid.For further information concerning phenotypically silent substitutionsin peptides and proteins see, e.g., Bowie J et al., Science 247: 1306-10(1990).

TABLE C Examples of Conservative Amino Acid Substitutions I II III IV VVI VII VIII IX X XI XII XIII XIV A D H C F N A C F A C A A D G E K I W QG M H C D C C E P Q R L Y S I P W F E D D G S N M T L Y G H G E K T V VH K N G P I N P H Q L Q S K R M R T N S R S V Q T T T R V S W P Y T

In the conservative substitution scheme in Table C, exemplaryconservative substitutions of amino acids are grouped by physicochemicalproperties—I: neutral, hydrophilic; II: acids and amides; III: basic;IV: hydrophobic; V: aromatic, bulky amino acids, VI hydrophilicuncharged, VII aliphatic uncharged, VIII non-polar uncharged, IXcycloalkenyl-associated, X hydrophobic, XI polar, XII small, XIIIturn-permitting, and XIV flexible. For example, conservative amino acidsubstitutions include the following: 1) S may be substituted for C; 2) Mor L may be substituted for F; 3) Y may be substituted for M; 4) Q or Emay be substituted for K; 5) N or Q may be substituted for H; and 6) Hmay be substituted for N.

Additional conservative amino acid substitutions include thefollowing: 1) S may be substituted for C; 2) M or L may be substitutedfor F; 3) Y may be substituted for M; 4) Q or E may be substituted forK; 5) N or Q may be substituted for H; and 6) H may be substituted forN.

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention may comprisefunctional fragments or variants of a polypeptide region of the presentinvention described herein that have, at most, 20, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid substitutions compared to a polypeptidesequence recited herein, as long as it (1) comprises at least oneembedded or inserted, heterologous T-cell epitope and at least one aminoacid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitoperegion provided in the Examples (see e.g. Tables 1-7 and/or 12), whereinthe disrupted amino acid does not overlap with the embedded or insertedepitope; (2) comprises at least one embedded or inserted, heterologousT-cell epitope and a disrupted furin-cleavage motif at thecarboxy-terminus of a Shiga toxin A1 fragment derived region; or (3)comprises a disrupted furin-cleavage motif at the carboxy-terminus of aShiga toxin A1 fragment derived region and comprises at least one aminoacid is disrupted in an endogenous, B-cell and/or CD4+ T-cell epitoperegion provided in the Examples (see e.g. Tables 1-7 and/or 12), whereinthe disrupted amino acid does not overlap with the disruptedfurin-cleavage motif. Variants of the Shiga toxin effector polypeptidesand cell-targeting molecules of the invention are within the scope ofthe present invention as a result of changing a polypeptide describedherein by altering one or more amino acid residues or deleting orinserting one or more amino acid residues, such as within the bindingregion or Shiga toxin effector polypeptide region, in order to achievedesired properties, such as changed cytotoxicity, changed cytostaticeffects, changed immunogenicity, and/or changed serum half-life. TheShiga toxin effector polypeptides and cell-targeting molecules of thepresent invention may further be with or without a signal sequence.

Accordingly, in certain embodiments, the Shiga toxin effectorpolypeptides of the present invention comprise or consists essentiallyof amino acid sequences having at least 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99%, overall sequence identity to anaturally occurring Shiga toxin A Subunit or fragment thereof, such as,e.g., Shiga toxin A Subunit, such as SLT-1A (SEQ ID NO:1), StxA (SEQ IDNO:2), and/or SLT-2A (SEQ ID NO:3), wherein the Shiga toxin effectorpolypeptide (1) comprises at least one embedded or inserted,heterologous T-cell epitope and at least one amino acid is disrupted inan endogenous, B-cell and/or CD4+ T-cell epitope region provided in theExamples (see e.g. Tables 1-7 and/or 12), and wherein the disruptedamino acid does not overlap with the embedded or inserted epitope; (2)comprises at least one embedded or inserted, heterologous T-cell epitopeand a disrupted furin-cleavage motif at the carboxy-terminus of a Shigatoxin A1 fragment derived region; or (3) comprises a disruptedfurin-cleavage motif at the carboxy-terminus of a Shiga toxin A1fragment derived region and comprises at least one amino acid isdisrupted in an endogenous, B-cell and/or CD4+ T-cell epitope regionprovided in the Examples (see e.g. Tables 1-7 and/or 12), and whereinthe disrupted amino acid does not overlap with the disruptedfurin-cleavage motif.

In certain embodiments of the Shiga toxin effector polypeptides of thepresent invention, one or more amino acid residues may be mutated,inserted, or deleted in order to increase the enzymatic activity of theShiga toxin effector polypeptide. In certain embodiments of the Shigatoxin effector polypeptides of the present invention, one or more aminoacid residues may be mutated or deleted in order to reduce or eliminatecatalytic and/or cytotoxic activity of the Shiga toxin effectorpolypeptide. For example, the catalytic and/or cytotoxic activity of theA Subunits of members of the Shiga toxin family may be diminished oreliminated by mutation or truncation.

The cytotoxicity of the A Subunits of members of the Shiga toxin familymay be altered, reduced, or eliminated by mutation and/or truncation.The positions labeled tyrosine-77, glutamate-167, arginine-170,tyrosine-114, and tryptophan-203 have been shown to be important for thecatalytic activity of Stx, Stx1, and Stx2 (Hovde C et al., Proc NatlAcad Sci USA 85: 2568-72 (1988); Deresiewicz R et al., Biochemistry 31:3272-80 (1992); Deresiewicz R et al., Mol Gen Genet 241: 467-73 (1993);Ohmura M et al., Microb Pathog 15: 169-76 (1993); Cao C et al.,Microbiol Immunol 38: 441-7 (1994); Suhan M, Hovde C, Infect Immun 66:5252-9 (1998)). Mutating both glutamate-167 and arginine-170 eliminatedthe enzymatic activity of Slt-I A1 in a cell-free ribosome inactivationassay (LaPointe P et al., J Biol Chem 280: 23310-18 (2005)). In anotherapproach using de novo expression of Slt-I A1 in the endoplasmicreticulum, mutating both glutamate-167 and arginine-170 eliminated Slt-IA1 fragment cytotoxicity at that expression level (LaPointe P et al., JBiol Chem 280: 23310-18 (2005)). A truncation analysis demonstrated thata fragment of StxA from residues 75 to 268 still retains significantenzymatic activity in vitro (Haddad J et al., J Bacteriol 175: 4970-8(1993)). A truncated fragment of Slt-I A1 containing residues 1-239displayed significant enzymatic activity in vitro and cytotoxicity by denovo expression in the cytosol (LaPointe P et al., J Biol Chem 280:23310-18 (2005)). Expression of a Slt-I A1 fragment truncated toresidues 1-239 in the endoplasmic reticulum was not cytotoxic because itcould not retrotranslocate to the cytosol (LaPointe P et al., J BiolChem 280: 23310-18 (2005)).

The most critical residues for enzymatic activity and/or cytotoxicity inthe Shiga toxin A Subunits were mapped to the followingresidue-positions: asparagine-75, tyrosine-77, tyrosine-114,glutamate-167, arginine-170, arginine-176, and tryptophan-203 amongothers (Di R et al., Toxicon 57: 525-39 (2011)). In particular, adouble-mutant construct of Stx2A containing glutamate-E167-to-lysine andarginine-176-to-lysine mutations was completely inactivated; whereas,many single mutations in Stx1 and Stx2 showed a 10-fold reduction incytotoxicity. Further, truncation of Stx1A to 1-239 or 1-240 reduced itscytotoxicity, and similarly, truncation of Stx2A to a conservedhydrophobic residue reduced its cytotoxicity.

The most critical residues for binding eukaryotic ribosomes and/oreukaryotic ribosome inhibition in the Shiga toxin A Subunit have beenmapped to the following residue-positions arginine-172, arginine-176,arginine-179, arginine-188, tyrosine-189, valine-191, and leucine-233among others (McCluskey A et al., PLoS One 7: e31191 (2012). However,certain modification may increase a Shiga toxin functional activityexhibited by a Shiga toxin effector polypeptide of the presentinvention. For example, mutating residue-position alanine-231 in Stx1Ato glutamate increased Stx1A's enzymatic activity in vitro (Suhan M,Hovde C, Infect Immun 66: 5252-9 (1998)).

In certain embodiments of Shiga toxin effector polypeptides of thepresent invention derived from SLT-1A (SEQ ID NO:1) or StxA (SEQ IDNO:2), the one or more amino acid residues mutated include substitutionof the asparagine at position 75, tyrosine at position 77, tyrosine atposition 114, glutamate at position 167, arginine at position 170,arginine at position 176, and/or substitution of the tryptophan atposition 203. Examples of such substitutions will be known to theskilled worker based on the prior art, such as asparagine at position 75to alanine, tyrosine at position 77 to serine, substitution of thetyrosine at position 114 to serine, substitution of the glutamateposition 167 to glutamate, substitution of the arginine at position 170to alanine, substitution of the arginine at position 176 to lysine,substitution of the tryptophan at position 203 to alanine, and/orsubstitution of the alanine at 231 with glutamate. Other mutations whicheither enhance or reduce Shiga toxin enzymatic activity and/orcytotoxicity are within the scope of the invention and may be determinedusing well known techniques and assays disclosed herein.

The Shiga toxin effector polypeptides and cell-targeting molecules ofthe present invention may optionally be conjugated to one or moreadditional agents, which may include therapeutic agents, diagnosticagents, and/or other additional exogenous materials known in the art,including such agents as described herein. In certain embodiments, theShiga toxin effector polypeptide or cell-targeting molecule of thepresent invention is PEGylated or albuminated, such as, e.g., to providede-immunization, disrupt furin-cleavage by masking the extended loopand/or the furin-cleavage motif at the carboxy-terminus of a Shiga toxinA1 fragment derived region, improve pharmacokinetic properties, and/orimprove immunogenicity (see e.g., Wang Q et al., Cancer Res 53: 4588-94(1993); Tsutsumi Y et al., Proc Natl Acad Sci USA 97: 8548-53 (2000);Buse J, El-Aneed A, Nanomed 5: 1237-60 (2010); Lim S et al., J ControlRelease 207-93 (2015)).

V. General Functions of the Cell-Targeting Molecules of the PresentInvention

The functional association of Shiga toxin effector polypeptides of thepresent invention with cell-targeting binding regions enables thecreation of cell-targeting molecules which selectively kill, inhibit thegrowth of, deliver exogenous material to, and/or detect specific celltypes. The properties of the Shiga toxin effector polypeptide of thepresent invention enable the creation of cell-targeting molecules withimproved therapeutic windows in chordates as compared to prior Shigatoxin effector polypeptides.

For certain embodiments, the cell-targeting molecule of the presentinvention provides, after administration to a chordate, one or more ofthe following: 1) potent and selective killing of targeted cells, e.g.,infected or malignant cells, at low administration doses, 2) linkagestability between the cell-targeting binding region and the Shiga toxineffector polypeptide region while the cell-targeting molecule is presentin extracellular spaces, 3) low levels of off-target cell deaths and/orunwanted tissue damage, and 4) cell-targeted delivery of heterologous,CD8+ T-cell epitopes for presentation by target cells in order toinitiate desirable, T-cell mediated, immune responses, such as, e.g.,the recruitment of CD8+ T-cells and the localized release of cytokinesat a tissue locus.

The Shiga toxin effector polypeptides and cell-targeting molecules ofthe present invention are useful in diverse applications involving,e.g., cell-killing; cell growth inhibition; intracellular, cargodelivery; biological information gathering; immune response stimulation,and/or remediation of a health condition. The Shiga toxin effectorpolypeptides of the present invention are useful as components ofvarious therapeutic and/or diagnostic molecules, such as, e.g.ligand-toxin fusions, immunotoxins, and/or immuno-conjugates. Thecell-targeting molecules of the present invention are useful astherapeutic and/or diagnostic molecules, such as, e.g., ascell-targeting, cytotoxic, therapeutic molecules; cell-targeting,nontoxic, delivery vehicles; and/or cell-targeting, diagnosticmolecules; for examples in applications involving the in vivo targetingof specific cell types for the diagnosis or treatment of a variety ofdiseases, including cancers, immune disorders, and microbial infections.

Depending on the embodiment, a Shiga toxin effector polypeptide orcell-targeting molecule of the present invention may have or provide oneor more of the following characteristics or functionalities: (1)de-immunization, (2) protease-cleavage resistance, (3) potentcytotoxicity at certain concentrations, (4) intracellular delivery of acargo consisting of an additional material (e.g. a heterologous, T-cellepitope), (4) selective cytotoxicity, (6) low off-target toxicity inmulticellular organisms at certain doses or dosages, (7) delivery of aheterologous, T-cell epitope to the MHC class I presentation pathway ofa target cell, and/or (8) stimulation of CD8+ T-cell immune response(s).Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are multi-functionalbecause the molecules have two or more of the characteristics orfunctionalities described herein. Certain further embodiments of thecell-targeting molecules of the present invention provide all of theaforementioned characteristics and functionalities in a single molecule.

The associating, coupling, and/or linking of a cell-targeting bindingregion(s) with a Shiga toxin effector polypeptide(s) of the presentinvention enables the engineering of cell-targeting molecules with Shigatoxin function(s) that can produce less adverse effects afteradministration at certain doses or dosages to a multicellular organismsuch as a mammal. Non-limiting examples of adverse effects includeoff-target toxicities, untargeted cytotoxicities, and/or unwanted immuneresponses. Certain embodiments of the Shiga toxin effector polypeptidesand cell-targeting molecules of the present invention are particularlyuseful in applications involving administration of a Shiga toxineffector polypeptide and/or cell-targeting molecule to a chordatebecause of functional properties, such as, e.g., de-immunization,reduced off-target toxicities, and/or targeted stimulation of desirableimmune responses such as via cell-surface presentation of acell-targeting molecule delivered, CD8+ T-cell epitope.

In certain embodiments, the cell-targeting molecules of the presentinvention are capable of binding extracellular target biomoleculesassociated with the cell surface of particular cell types and enteringthose cells. Once internalized within a targeted cell type, certainembodiments of the cell-targeting molecules of the invention are capableof routing an enzymatically active, cytotoxic, Shiga toxin effectorpolypeptide fragment into the cytosol of the target cell and eventuallykilling the cell. Alternatively, nontoxic or reduced-toxicity variantsof the cell-targeting molecules of the present invention may be used todeliver additional exogenous materials into target cells, such asepitopes, peptides, proteins, polynucleotides, and detection promotingagents. This system is modular, in that any number of diverse bindingregions can be used to target a Shiga toxin effector polypeptide of thepresent invention to various, diverse cell types.

A. De-Immunization for Applications Involving Administration to aChordate

The de-immunization of the Shiga toxin effector polypeptides of thepresent invention is accomplished by engineering disruptions of one ormore, endogenous, B-cell and/or CD4+ T-cell epitopes regions of a Shigatoxin A Subunit or Shiga toxin effector polypeptide, including viamutation and/or truncation or via the conjugation of a covalently-linkedchemical structure. Because B-cell epitopes often coincide or overlapwith epitopes of mature CD4+ T-cells, the disruption of an endogenous,B-cell epitope region often simultaneously disrupts an endogenous, CD4+T-cell epitope or vice versa.

Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are de-immunized withrespect to one or more B-cell and/or CD4+ T-cell epitopes meaning thatthese molecules exhibit reduced antigenic and/or immunogenic potentialas compared to prior, Shiga toxin effector polypeptides andcell-targeting molecules lacking identical disruptions to the sameB-cell and/or CD4+ T-cell epitope or epitope regions and/or lacking anydisruption to the same B-cell and/or CD4+ T-cell epitope(s) or epitoperegion(s). Certain further embodiments exhibit potent if not wild-typelevels of Shiga toxin A Subunit catalytic domain dependent cytotoxicitydespite the presence of multiple mutations providing the de-immunizedproperty. The de-immunized, Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are useful forapplications involving the parenteral administration of a Shiga toxineffector polypeptide and/or cell-targeting molecule to a chordate suchas, e.g., a mammal, amphibian, bird, fish, reptiles, or shark, becauseof the reduced likelihood of producing undesirable immune responsesinvoked by the administrated molecule.

The various de-immunized, Shiga toxin effector polypeptides of thepresent invention might differ in their antigenicity profiles whenadministered to various chordate species, but all the de-immunizedpolypeptides of the invention exhibit reduced antigenicity and/orimmunogenicity in at least one organism as measured by at least onequantitative assay. In particular, certain embodiments of thecell-targeting molecules of the present invention are de-immunized withrespect to a mammalian recipient, such as, e.g., the molecule invokeslower quantities and/or frequencies of “anti-cell-targeting molecule”antibodies when administered to that mammal as compared to a referencemolecule (e.g. a related cell-targeting molecule comprising a wild-typeShiga toxin A1 fragment). In addition, Shiga toxin effector polypeptidesof the present invention having disruptions of multiple, endogenous,epitope regions are expected to more greatly reduced the probability ofthe occurrence of undesirable immune responses in a chordate recipientof such a polypeptide.

For certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, the de-immunizationproperty(ies) is a result of the structural change(s) which include thedisrupted furin-cleavage motif at the carboxy-terminus of a Shiga toxinA1 fragment derived region.

For certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, the de-immunizationproperty(ies) is a result of the structural change(s) which include theembedding and/or inserting of a T-cell epitope which disrupts anendogenous, B-cell and/or CD4+ T-cell epitope region.

For certain embodiments, the desired biological function(s) of theparental, Shiga toxin polypeptide from which the de-immunized, Shigatoxin effector polypeptide was derived are preserved, such as, e.g., theShiga toxin A Subunit functions of promoting cellular internalization,directing intracellular routing, and potent cytotoxicity. Preservationrefers to the retention of a minimal level of activity as describedherein.

B. Reduced Protease-Cleavage Sensitivity

Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention exhibit reducedprotease-cleavage sensitivity as compared to related moleculescomprising wild-type, Shiga toxin A1 fragment regions. Certain furtherembodiments exhibit potent if not optimal, Shiga toxin A Subunitcatalytic domain dependent cytotoxicity despite this reducedprotease-cleavage sensitivity and lack of a canonical furin-cleavageevent within an intoxicated cell.

Certain embodiments of the protease-cleavage resistant, cell-targetingmolecules of the present invention (i.e. a cell-targeting moleculecomprising a Shiga toxin effector polypeptide comprising a disruptedfurin-cleavage motif at the carboxy-terminus of its Shiga toxin A1fragment region) exhibit improved in vivo tolerability as compared torelated molecules comprising a wild-type, Shiga toxin A1 fragmentregion. Certain further embodiments exhibit potent if not optimal, Shigatoxin A Subunit catalytic domain dependent cytotoxicity despite thisreduced protease-cleavage sensitivity and lack of a canonicalfurin-cleavage event within an intoxicated cell.

Previously, it was believed that cytotoxic, Shiga toxin A Subunitconstructs comprising Shiga toxin A1 fragment catalytic regions mustmaintain or somehow compensate for the naturally occurring proteolyticprocessing by furin within intoxicated cells in order to preserve theShiga toxin's natural adaptations for efficient and potent cytotoxicity.It was unexpectedly discovered that the furin cleavage event was notrequired for potent cytotoxicity because potent Shiga toxin cytotoxicityat the level of a wild-type Shiga toxin control construct was achievedin the absence of any furin cleavage event at the carboxy-terminus ofthe Shiga toxin A1 fragment despite the presence of a carboxy-terminalmoiety (see Examples, infra and WO 2015/191764). The lack of afurin-cleavage event within the intoxicated cell may prevent theefficient liberation of a Shiga toxin A1 fragment-like region and, thus,result in the continued linkage of a relatively large moiety (e.g.greater than 28 kDa in size) to the Shiga toxin A1 fragment region.However despite this possibility, potent, Shiga toxin cytotoxicity wasachieved with furin-cleavage deficient constructs comprising a Shigatoxin effector polypeptide region and lacking any known compensatoryfeature(s), such as, e.g., providing intracellular cleavage proximal tothe carboxy-terminus of a Shiga toxin A1 fragment derived region (seeExamples, infra; WO 2015/191764).

This suggests that the persistence and/or inefficient release of arelatively large, molecular moiety linked to the A1 fragment region didnot necessarily reduce the potency of Shiga toxin cytotoxicity. This wassurprising because the optimal Shiga toxin intoxication process wasthought to require liberation of the Shiga toxin A1 fragments from allother large molecular moieties to efficiently retrotranslocate liberatedA1 fragments from the endoplasmic reticulum to the cytosol where the A1fragments can form an enzymatically active structure that catalyticallyinactivates the intoxicated cell's ribosomes. In particular, thepersistence and/or inefficient release of a relatively large molecularmoiety covering the carboxy-terminus of the Shiga toxin A1 fragment wasexpected to interfere with the Shiga toxin A1 fragment's naturalmechanism of efficiently gaining access to the cytosol, which involvesthe exposure of the A1 fragment's, hydrophobic, carboxy-terminal domainand recognition of this domain by the ERAD system (see Di R et al.,Toxicon 57: 525-39 (2011); Li S et al., PLoS One 7: e41119 (2012)).

The lack of an intoxicated-cell-mediated, furin-cleavage event for amolecule comprising a Shiga toxin A Subunit derivative may behypothetically compensated for. Non-limiting examples of potential,compensatory approaches include 1) terminating one carboxy-terminus ofthe construct with the carboxy-terminus of a Shiga toxin A1fragment-like polypeptide region, 2) producing the Shiga toxin derivedconstruct such that the Shiga toxin A Subunit polypeptide is alreadynicked near the carboxy-terminus of its Shiga toxin A1 fragment-likepolypeptide, 3) engineering a heterologous and/or ectopic protease sitethat can functionally substitute for the lack of the native, Shigatoxin, furin-cleavage event, and 4) a combination of approach 3 and 4.

In the first approach, the carboxy-terminus of the Shiga toxin A1fragment-like polypeptide is not covered by any carboxy-terminal moiety,and, thus, the carboxy-terminus of the Shiga toxin A1 fragment-likepolypeptide is permanently exposed for recognition by the ERAD machineryin the endoplasmic reticulum. In the last three approaches, the Shigatoxin A1 fragment-like polypeptide can be designed to intracellularlydissociate from one or more other components of the construct by thetime the molecule reaches the endoplasmic reticulum of an intoxicatedcell such that in the endoplasmic reticulum the carboxy-terminus of theShiga toxin A1 fragment-like polypeptide becomes exposed for recognitionby the ERAD machinery. For example, a cytotoxic molecule comprising aShiga toxin A Subunit effector polypeptide could be pretreated with aprotease to nick the polypeptide region near the carboxy terminus of theA1 fragment-like region prior to contacting a target cell.Alternatively, the cytotoxic molecule could be engineered to comprise aprotease site which is cleaved by an intracellular protease of thetarget cell.

These hypothetical approaches for designing Shiga toxin A Subuniteffector polypeptides which compensate for the lack of anintoxicated-cell-mediated, furin-cleavage event may significantly alterthe efficiency and potency of cytotoxicity as compared to a wild-typeShiga holotoxin or Shiga toxin A Subunit construct comprising onlywild-type sequences which include the optimal, naturally occurring,furin-cleavage site. For example, currently no compensatory approachrelying on a target cell endoprotease other than furin is known whichcan provide fully compensatory cytotoxicity equivalent to furin cleavageand alternative cellular proteases to furin like calpains have beenshown to be less efficient in facilitating Shiga toxin cytotoxicity(Garred O et al., Exp Cell Res 218: 39-49 (1995); Garred O et al., JBiol Chem 270: 10817-21 (1995); Kurmanova A et al., Biochem Biophys ResCommun 357: 144-9 (2007)).

The present invention provides furin-cleavage resistant Shiga toxin ASubunit effector polypeptides which are potently cytotoxic, whether dueto compensation for a lack of a furin cleavage event within theintoxicated cell or due to some unexplained reason. Certaincell-targeting molecules of the present invention are at least asefficiently and potently cytotoxic as cell-targeting moleculescomprising protease-cleavage sensitive, wild-type Shiga toxin effectorpolypeptide regions (see Examples, infra).

C. Improved Stability and In Vivo Tolerability

In certain embodiments, the molecules of the present invention (e.g.cell-targeting molecules of the invention) exhibit increased stabilityand/or improved in vivo tolerability as compared to more furin-cleavagesensitive analogs and/or less de-immunized analogs (an analog being aclosely related molecule lacking one or more structural features of thepresent invention).

The increased stability of a cell-targeting molecule compared to areference molecule can be exhibited in vitro and/or in vivo. Thestability of a therapeutic or diagnostic molecule over time is animportant feature and can affect for which applications the molecule maybe practically employed. Molecular stability includes in vitro and invivo, such as, e.g., stability within an organism after administrationand during storage over a range of temperatures and concentrations. Forcertain immunotoxins or ligand-toxin fusions, the stability of thelinkage between the toxin and other components can affect the amount ofnon-specific toxicity caused by the presence and/or quantity ofuntargeted toxin over time within the organism.

Certain cell-targeting molecules of the present invention exhibitreduced non-specific toxicity in vivo, manifested as increased in vivotolerability as compared to more protease-cleavage sensitive variants.In vivo tolerability can be determined by the skilled worker usingtechniques known in the art and/or described herein. In addition toassessing in vivo tolerability using mortality, signs of morbidity maybe used for assessing in vivo tolerability, such as, e.g., aspects ofbody weight, physical appearance, measureable clinical signs, unprovokedbehavior, and responses to external stimuli (see e.g. Morton D,Griffiths P, Vet Rec 116: 431-43 (1985); Montgomery C, Cancer Bull 42:230-7 (1990); Ullman-Culleré M, Foltz C, Lab Anim Sci 49: 319-23 (1999);Clingerman K, Summers L, J Am Assoc Lab Anim Sci 51: 31-6 (2012)).Euthanasia may be used in response to signs of morbidity and/ormorbundity and, thus, create a mortality time-point. For example, adecrease in body weight of 15-20% in 2-3 days can be used as a sign ofmorbidity in rodents and as a justification for euthanization (see e.g.Institute of Laboratory Animal Research 2011. Guide for the care and useof laboratory animals, 8th ed., Washington, D.C., U.S.: NationalAcademies Press).

The improved in vivo tolerability observed for exemplary, cell-targetingmolecules of the present invention as compared to more furin-cleavagesensitive analogs suggests that much higher doses of thesecell-targeting molecules of the invention may be safely administered tomammals as compared to the doses of related molecules comprising afurin-cleavage sensitive, Shiga toxin effector polypeptide region.Certain cell-targeting molecules of the invention might exhibit reducednon-specific toxicity as compared to more protease sensitive variantsbecause the protease resistance serves to protect and preserve thelinkage between the Shiga toxin effector component and thecell-targeting moiety component.

In addition, in vivo tolerability for cell-targeting molecules of thepresent invention may be related to the de-immunization properties of agiven cell-targeting molecule. Thus, higher doses of such de-immunized,cell-targeting molecules of the invention may be safely administered tomammals as compared to the doses of related molecules comprising an“un-de-immunized” or less de-immunized, Shiga toxin effector polypeptide(e.g. a wild-type Shiga toxin A1 fragment).

In addition, certain molecules of the invention exhibit increasedhalf-lives, both in vitro and/or in vivo, as compared to moreprotease-cleavage sensitive variants. Molecular stability can be assayedby determining the half-life of a molecule of interest with regard tothe association of its components. Certain embodiments of the moleculesof the invention will have longer half-lives as compared tofurin-cleavage sensitive variants, especially with regard to thecontinued association of the Shiga toxin effector polypeptide componentand one or more other components. For example, certain embodiments ofthe molecules of the invention will have longer half-lives with regardto the continued association of the Shiga toxin effector polypeptidecomponent and another component, e.g. a cell-targeting binding region,as compared to a furin-cleavage sensitive variant wherein thefurin-cleavage sensitive site(s) lies between those two components.

D. Cell-Kill Via Shiga Toxin a Subunit Cytotoxicity

Certain embodiments of the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are cytotoxic. Certainfurther embodiments of the cell-targeting molecules of the presentinvention are cytotoxic only due to the presence of one or more Shigatoxin effector polypeptide components. The A Subunits of members of theShiga toxin family each comprise an enzymatically active polypeptideregion capable of killing a eukaryotic cell once in the cell's cytosol.Because members of the Shiga toxin family are adapted to killingeukaryotic cells, molecules derived from Shiga toxins, such as, e.g.,molecules comprising certain embodiments of the Shiga toxin effectorpolypeptides of the present invention can exhibit potent cell-killactivities.

For certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the binding region of thecell-targeting molecule (e.g. a target positive cell), thecell-targeting molecule is capable of causing death of the cell. Forcertain further embodiments, the CD₅₀ value of the cell-targetingmolecule is less than 5, 2.5, 1, 0.5, or 0.25 nM, which is vastly morepotent than an untargeted, wild-type, Shiga toxin effector polypeptide(e.g. SEQ ID NO. 4).

Cell-kill may be accomplished using a molecule of the present inventionunder varied conditions of target cells, such as, e.g., an ex vivomanipulated target cell, a target cell cultured in vitro, a target cellwithin a tissue sample cultured in vitro, or a target cell in an in vivosetting like within a multicellular organism.

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention comprise (1) ade-immunized, Shiga toxin effector sub-region, (2) a protease-cleavageresistant region near the carboxy-terminus of a Shiga toxin A1 fragmentderived region, (3) a carboxy-terminal, endoplasmic reticulumretention/retrieval signal motif, and/or (4) a heterologous, T-cellepitope embedded or inserted region; however, for certain furtherembodiments, these structural modifications do not significantly alterthe potency of Shiga toxin cytotoxicity as compared to a referencemolecules comprising a wild-type Shiga toxin A Subunit polypeptide, suchas, e.g., a wild-type Shiga toxin A1 fragment. Thus, Shiga toxineffector polypeptides and cell-targeting molecules of the presentinvention which are de-immunized, protease cleavage resistant, and/orcarrying embedded or inserted, heterologous, epitopes can maintainpotent cytotoxicity while providing one or more various otherfunctionalities or properties.

Already cytotoxic cell-targeting molecules comprising Shiga toxineffector polypeptides may be engineered by the skilled worker using theinformation and methods provided herein to be more cytotoxic and/or tohave redundant, backup cytotoxicities operating via completely differentmechanisms. These multiple cytotoxic mechanisms may complement eachother by their diversity of functions (such as by providing potentkilling via two mechanisms of cell-killing, direct and indirect, as wellas mechanisms of immuno-stimulation to the local area), redundantlybackup each other (such as by providing one cell-killing mechanism inthe absence of the other mechanisms-like if a target cell is resistantto or acquires some immunity to a subset of previously activemechanisms), and/or protect against developed resistance (by limitingresistance to the less probable situation of the malignant or infectedcell blocking multiple, different cell-killing mechanismssimultaneously).

E. Delivery of a T-Cell Epitope for MHC Class I Presentation on a CellSurface

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention comprise a T-cellepitope, which enables the engineering of “T-cell epitope delivering”molecules with virtually unlimited choices of epitope-peptide cargos fordelivery and cell-surface presentation by a nucleated, chordate cell.For certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention are each capable ofdelivering one or more T-cell epitopes, associated with the Shiga toxineffector polypeptides and/or cell-targeting molecules, to the proteasomeof a cell.

The delivered T-cell epitope are then proteolytic processed andpresented by the MHC class I pathway on the surface of the cell. Byengineering MHC class I epitopes into cell-targeting molecules, thetargeted delivery and presentation of immuno-stimulatory antigens may beaccomplished in order to harness and direct a beneficial function(s) ofa chordate immune system.

For certain embodiments, the Shiga toxin effector polypeptide orcell-targeting molecule of the present invention is capable ofdelivering a T-cell epitope to a MHC class I molecule of a cell forcell-surface presentation. In certain embodiments, the Shiga toxineffector polypeptide or cell-targeting molecule of the present inventioncomprises a heterologous, T-cell epitope, whether as an additionalexogenous material or embedded or inserted within a Shiga toxin effectorpolypeptide. For certain further embodiments, the Shiga toxin effectorpolypeptide or cell-targeting molecule of the present invention iscapable of delivering an embedded or inserted T-cell epitope to a MHCclass I molecule for cell-surface presentation.

For certain embodiments, the Shiga toxin effector polypeptide of thepresent invention is capable of delivering a T-cell epitope, which isembedded or inserted in the Shiga toxin effector polypeptide, to a MHCclass I molecule of a cell in which the Shiga toxin effector polypeptideis present for presentation of the T-cell epitope by the MHC class Imolecule on a surface of the cell. For certain further embodiments, theT-cell epitope is a heterologous, T-cell epitope. For certain furtherembodiments, the T-cell epitope functions as CD8+ T-cell epitope,whether already known or identified in the future using methods whichare currently routine to the skilled worker.

For certain embodiments, the cell-targeting molecule of the presentinvention is capable of delivering a T-cell epitope, which is associatedwith the cell-targeting molecule, to a MHC class I molecule of a cellfor presentation of the T-cell epitope by the MHC class I molecule on asurface of the cell. For certain further embodiments, the T-cell epitopeis a heterologous, T-cell epitope which is embedded or inserted in theShiga toxin effector polypeptide. For certain further embodiments, theT-cell epitope functions as CD8+ T-cell epitope, whether already knownor identified in the future using methods which are currently routine tothe skilled worker.

For certain embodiments, upon contacting a cell with the cell-targetingmolecule of the present invention, the cell-targeting molecule iscapable of delivering a T-cell epitope-peptide, which is associated withthe cell-targeting molecule, to a MHC class I molecule of the cell forpresentation of the T-cell epitope-peptide by the MHC class I moleculeon a surface of the cell. For certain further embodiments, the T-cellepitope-peptide is a heterologous epitope which is embedded or insertedin a Shiga toxin effector polypeptide. For certain further embodiments,the T-cell epitope-peptide functions as CD8+ T-cell epitope, whetheralready known or identified in the future using methods which arecurrently routine to the skilled worker.

The addition of a heterologous epitope into or presence of aheterologous epitope in a cell-targeting molecule of the presentinvention, whether as an additional exogenous material or embedded orinserted within a Shiga toxin effector polypeptide, enables methods ofusing such cell-targeting molecules for the cell-targeted delivery of achosen epitope for cell-surface presentation by a nucleated, target cellwithin a chordate.

One function of certain, CD8+ T-cell hyper-immunized, Shiga toxineffector polypeptides and cell-targeting molecules of the presentinvention is the delivery of one or more T-cell epitope-peptides to aMHC class I molecule for MHC class I presentation by a cell. Delivery ofexogenous, T-cell epitope-peptides to the MHC class I system of a targetcell can be used to induce the target cell to present the T-cellepitope-peptide in association with MHC class I molecules on the cellsurface, which subsequently leads to the activation of CD8+ effectorT-cells to attack the target cell.

The skilled worker, using techniques known in the art, can associate,couple, and/or link certain, Shiga toxin effector polypeptides of thepresent invention to various other cell-targeting binding region tocreate cell-targeting molecules of the present invention which targetspecific, extracellular, target biomolecules physically coupled to cellsand promote target-cell internalization of these cell-targetingmolecules. All nucleated vertebrate cells are believed to be capable ofpresenting intracellular epitopes using the MHC class I system. Thus,extracellular target biomolecules of the cell-targeting molecules of theinvention may in principle target any nucleated vertebrate cell forT-cell epitope delivery to a MHC class I presentation pathway of such acell.

The epitope-delivering functions of the Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention canbe detected and monitored by a variety of standard methods known in theart to the skilled worker and/or described herein. For example, theability of cell-targeting molecules of the present invention to delivera T-cell epitope-peptide and drive presentation of the epitope-peptideby the MHC class I system of target cells may be investigated usingvarious in vitro and in vivo assays, including, e.g., the directdetection/visualization of MHC class I/peptide complexes, measurement ofbinding affinities for the heterologous, T-cell epitope-peptide to MHCclass I molecules, and/or measurement of functional consequences of MHCclass I-peptide complex presentation on target cells by monitoringcytotoxic T-lymphocyte (CTL) responses (see e.g. Examples, infra).

Certain assays to monitor this function of the polypeptides andmolecules of the present invention involve the direct detection of aspecific MHC class I/peptide antigen complex in vitro or ex vivo. Commonmethods for direct visualization and quantitation of peptide-MHC class Icomplexes involve various immuno-detection reagents known to the skilledworker. For example, specific monoclonal antibodies can be developed torecognize a particular MHC/class I/peptide antigen complex. Similarly,soluble, multimeric T cell receptors, such as the TCR-STAR reagents(Altor Bioscience Corp., Mirmar, Fla., U.S.) can be used to directlyvisualize or quantitate specific MHC I/antigen complexes (Zhu X et al.,J Immunol 176: 3223-32 (2006)). These specific mAbs or soluble,multimeric T-cell receptors may be used with various detection methods,including, e.g. immunohistochemistry, flow cytometry, and enzyme-linkedimmuno assay (ELISA).

An alternative method for direct identification and quantification ofMHC I/peptide complexes involves mass spectrometry analyses, such as,e.g., the ProPresent Antigen Presentation Assay (ProImmune, Inc.,Sarasota, Fla., U.S.) in which peptide-MCH class I complexes areextracted from the surfaces of cells, then the peptides are purified andidentified by sequencing mass spectrometry (Falk K et al., Nature 351:290-6 (1991)).

In certain assays to monitor the T-cell epitope delivery and MHC class Ipresentation function of the polypeptides and molecules of the presentinvention involve computational and/or experimental methods to monitorMHC class I and peptide binding and stability. Several software programsare available for use by the skilled worker for predicting the bindingresponses of peptides to MHC class I alleles, such as, e.g., The ImmuneEpitope Database and Analysis Resource (IEDB) Analysis Resource MHC-Ibinding prediction Consensus tool (Kim Y et al., Nucleic Acid Res 40:W525-30 (2012). Several experimental assays have been routinely applied,such as, e.g., cell surface binding assays and/or surface plasmonresonance assays to quantify and/or compare binding kinetics (Miles K etal., Mol Immunol 48: 728-32 (2011)). Additionally, other MHC-peptidebinding assays based on a measure of the ability of a peptide tostabilize the ternary MHC-peptide complex for a given MHC class Iallele, as a comparison to known controls, have been developed (e.g.,MHC-peptide binding assay from ProImmmune, Inc.).

Alternatively, measurements of the consequence of MHC class I/peptideantigen complex presentation on the cell surface can be performed bymonitoring the cytotoxic T-cell (CTL) response to the specific complex.These measurements by include direct labeling of the CTLs with MHC classI tetramer or pentamer reagents. Tetramers or pentamers bind directly toT cell receptors of a particular specificity, determined by the MajorHistocompatibility Complex (MHC) allele and peptide complex.Additionally, the quantification of released cytokines, such asinterferon gamma or interleukins by ELISA or enzyme-linked immunospot(ELIspot) is commonly assayed to identify specific CTL responses. Thecytotoxic capacity of CTL can be measured using a number of assays,including the classical 51 Chromium (Cr) release assay or alternativenon-radioactive cytotoxicity assays (e.g., CytoTox96® non-radioactivekits and CellTox™ CellTiter-GLO® kits available from Promega Corp.,Madison, Wis., U.S.), Granzyme B ELISpot, Caspase Activity Assays orLAMP-1 translocation flow cytometric assays. To specifically monitor thekilling of target cells, carboxyfluorescein diacetate succinimidyl ester(CFSE) can be used to easily and quickly label a cell population ofinterest for in vitro or in vivo investigation to monitor killing ofepitope specific CSFE labeled target cells (Durward M et al., J Vis Exp45 pii 2250 (2010)).

In vivo responses to MHC class I presentation can be followed byadministering a MHC class I/antigen promoting agent (e.g., a peptide,protein or inactivated/attenuated virus vaccine) followed by challengewith an active agent (e.g. a virus) and monitoring responses to thatagent, typically in comparison with unvaccinated controls. Ex vivosamples can be monitored for CTL activity with methods similar to thosedescribed previously (e.g. CTL cytotoxicity assays and quantification ofcytokine release).

HLA-A, HLA-B, and/or HLA-C molecules are isolated from the intoxicatedcells after lysis using immune affinity (e.g., an anti-MHC antibody“pulldown” purification) and the associated peptides (i.e., the peptidespresented by the isolated MHC molecules) are recovered from the purifiedcomplexes. The recovered peptides are analyzed by sequencing massspectrometry. The mass spectrometry data is compared against a proteindatabase library consisting of the sequence of the exogenous (non-self)peptide (T-cell epitope X) and the international protein index forhumans (representing “self” or non-immunogenic peptides). The peptidesare ranked by significance according to a probability database. Alldetected antigenic (non-self) peptide sequences are listed. The data isverified by searching against a scrambled decoy database to reduce falsehits (see e.g. Ma B, Johnson R, Mol Cell Proteomics 11: O111.014902(2012)). The results will demonstrate that peptides from the T-cellepitope X are presented in MHC complexes on the surface of intoxicatedtarget cells.

The set of presented peptide-antigen-MHC complexes can vary betweencells due to the antigen-specific HLA molecules expressed. T-cells canthen recognize specific peptide-antigen-MHC complexes displayed on acell surface using different TCR molecules with differentantigen-specificities.

Because multiple T-cell epitopes may be delivered by a cell-targetingmolecule of the invention, such as, e.g., by embedding two or moredifferent T-cell epitopes in a single proteasome delivering effectorpolypeptide, a single cell-targeting molecule of the invention may beeffective chordates of the same species with different MHC classvariants, such as, e.g., in humans with different HLA alleles. This mayallow for the combining within a single molecule of different T-cellepitopes with different effectiveness in different sub-populations ofsubjects based on MHC complex protein diversity and polymorphisms. Forexample, human MHC complex proteins, HLA proteins, vary among humansbased on genetic ancestry, e.g. African (sub-Saharan), Amerindian,Caucasiod, Mongoloid, New Guinean and Australian, or Pacific islander.

The applications involving the T-cell epitope delivering polypeptidesand molecules of the present invention are vast. Every nucleated cell ina mammalian organism may be capable of MHC class I pathway presentationof immunogenic, T-cell epitope-peptides on their cell outer surfacescomplexed to MHC class I molecules. In addition, the sensitivity ofT-cell epitope recognition is so exquisite that only a few MIC-I peptidecomplexes are required to be presented to result in an immune response,e.g., even presentation of a single complex can be sufficient forrecognition by an effector T-cell (Sykulev Y et al., Immunity 4: 565-71(1996)).

The activation of T-cell responses are desired characteristics ofcertain anti-cancer, anti-neoplastic, anti-tumor, and/or anti-microbialbiologic drugs to stimulate the patient's own immune system towardtargeted cells. Activation of a robust and strong T-cell response isalso a desired characteristic of many vaccines. The presentation of aT-cell epitope by a target cell within an organism can lead to theactivation of robust immune responses to a target cell and/or itsgeneral locale within an organism. Thus, the targeted delivery of aT-cell epitope for presentation may be utilized for as a mechanism foractivating T-cell responses during a therapeutic regime.

The presentation of a T-cell immunogenic epitope-peptide by the MHCclass I system targets the presenting cell for killing by CTL-mediatedlysis and also triggers immune stimulation in the localmicroenvironment. By engineering immunogenic epitope sequences withinShiga toxin effector polypeptide components of target-cell-internalizingtherapeutic molecules, the targeted delivery and presentation ofimmuno-stimulatory antigens may be accomplished. The presentation ofimmuno-stimulatory non-self antigens, such as e.g. known viral antigenswith high immunogenicity, by target cells signals to other immune cellsto destroy the target cells as well as to recruit more immune cells tothe area.

The presentation of an immunogenic, T-cell epitope-peptide by the MHCclass I complex targets the presenting cell for killing by CTL-mediatedcytolysis. The presentation by targeted cells of immuno-stimulatorynon-self antigens, such as, e.g., known viral epitope-peptides with highimmunogenicity, can signal to other immune cells to destroy the targetcells and recruit more immune cells to the target cell site within achordate.

Thus, already cytotoxic molecules, such as e.g. therapeutic orpotentially therapeutic molecules comprising Shiga toxin effectorpolypeptides, may be engineered using methods of the present inventioninto more cytotoxic molecules and/or to have an additional cytotoxicmechanism operating via delivery of a T-cell epitope, presentation, andstimulation of effector T-cells. These multiple cytotoxic mechanisms maycomplement each other (such as by providing both directtarget-cell-killing and indirect (CTL-mediated) cell-killing,redundantly backup each other (such as by providing one mechanism ofcell-killing in the absence of the other), and/or protect against thedevelopment of therapeutic resistance (by limiting resistance to theless probable situation of the malignant or infected cell evolving toblock two different cell-killing mechanisms simultaneously).

In addition, a cytotoxic molecule comprising a Shiga toxin effectorpolypeptide region that exhibits catalytic-based cytotoxicity may beengineered by the skilled worker using routine methods intoenzymatically inactive variants. For example, the cytotoxic Shiga toxineffector polypeptide component of a cytotoxic molecule may be conferredwith reduced activity and/or rendered inactive by the introduction ofone or mutations and/or truncations such that the resulting molecule canstill be cytotoxic via its ability to deliver a T-cell epitope to theMHC class I system of a target cell and subsequent presentation to thesurface of the target cell. In another example, a T-cell epitope may beinserted or embedded into a Shiga toxin effector polypeptide such thatthe Shiga toxin effector polypeptide is inactivated by the added epitope(see e.g. WO 2015/113007). This approach removes one cytotoxic mechanismwhile retaining or adding another and may also provide a moleculecapable of exhibiting immuno-stimulation to the local area of a targetcell(s) within an organism via delivered T-cell epitope presentation or“antigen seeding.” Furthermore, non-cytotoxic variants of thecell-targeting molecules of the present invention which compriseembedded or inserted, heterologous, T-cell epitopes may be useful inapplications involving immune-stimulation within a chordate and/orlabeling of target cells within a chordate with MHC class I moleculedisplayed epitopes.

The ability to deliver a T-cell epitope of certain Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention maybe accomplished under varied conditions and in the presence ofnon-targeted bystander cells, such as, e.g., an ex vivo manipulatedtarget cell, a target cell cultured in vitro, a target cell within atissue sample cultured in vitro, or a target cell in an in vivo settinglike within a multicellular organism.

F. Cell-Kill Via Targeted Cytotoxicity and/or Engagement of CytotoxicT-Cells

For certain embodiments, the cell-targeting molecule of the presentinvention can provide 1) delivery of a T-cell epitope for MHC class Ipresentation by a target cell and/or 2) potent cytotoxicity. For certainembodiments of the cell-targeting molecules of the present invention,upon contacting a cell physically coupled with an extracellular targetbiomolecule of the cell-targeting binding region, the cell-targetingmolecule of the invention is capable of causing death of the cell. Themechanism of cell-kill may be direct, e.g. via the enzymatic activity ofa toxin effector polypeptide region, or indirect via CTL-mediatedcytolysis.

1. Indirect Cell-Kill Via T-Cell Epitope Delivery and MHC Class IPresentation

Certain embodiments of the cell-targeting molecules of the presentinvention are cytotoxic because they comprise a CD8+ T-cell epitopecapable of being delivered to the MHC class I presentation pathway of atarget cell and presented on a cellular surface of the target cell. Forexample, T-cell epitope delivering, CD8+ T-cell hyper-immunized, Shigatoxin effector polypeptides of the present invention, with or withoutendogenous epitope de-immunization, may be used as components ofcell-targeting molecules for applications involving indirectcell-killing.

In certain embodiments of the cell-targeting molecules of the presentinvention, upon contacting a cell physically coupled with anextracellular target biomolecule of the cell-targeting binding region,the cell-targeting molecule of the invention is capable of indirectlycausing the death of the cell, such as, e.g., via the presentation ofone or more T-cell epitopes by the target cell and the subsequentrecruitment of CTLs which kill the target cell.

The presentation of an antigenic peptide complexed with a MHC class Imolecule by a cell sensitizes the presenting cell to targeted killing bycytotoxic T-cells (CTLs) via the induction of apoptosis, lysis, and/ornecrosis. In addition, the CTLs which recognize the target cell mayrelease immuno-stimulatory cytokines, such as, e.g., interferon gamma(IFN-gamma), tumor necrosis factor alpha (TNF), macrophage inflammatoryprotein-1 beta (MIP-1beta), and interleukins such as IL-17, TL-4, andIL-22. Furthermore, CTLs activated by recognition of a presented epitopemay indiscriminately kill other cells proximal to the presenting cellregardless of the peptide-MHC class I complex repertoire presented bythose proximal cells (Wiedemann A et al., Proc Natl Acad Sci USA 103:10985-90 (2006)).

Because of MHC allele diversity within different species, acell-targeting molecule of the present invention comprising only asingle epitope may exhibit varied effectiveness to different patients orsubjects of the same species. However, certain embodiments of thecell-targeting molecules of the present invention may each comprisemultiple, T-cell epitopes that are capable of being delivered to the MHCclass I system of a target cell simultaneously. Thus, for certainembodiments of the cell-targeting molecules of the present invention, acell-targeting molecule is used to treat different subjects withconsiderable differences in their MHC molecules' epitope-peptide bindingaffinities (i.e. considerable differences in their MHC alleles and/orMHC genotypes). In addition, certain embodiments of the cell-targetingmolecules of the present invention reduce or prevent target celladaptations to escape killing (e.g. a target cancer cell mutating toescape therapeutic effectiveness or “mutant escape”) by using multiplecell-killing mechanisms simultaneously (e.g. direct killing and indirectkilling via multiple different T-cell epitopes simultaneously).

2. Direct Cell-Kill Via Cell-Targeted, Shiga Toxin Cytotoxicity

Certain embodiments of the cell-targeting molecules of the presentinvention are cytotoxic because they comprise a catalytically active,Shiga toxin effector polypeptide and regardless of the presence of animmunogenic, CD8+ T-cell epitope in the molecule. For example, CD8+T-cell hyper-immunized, Shiga toxin effector polypeptides of the presentinvention, with or without endogenous epitope de-immunization, may beused as components of cell-targeting molecules for applicationsinvolving direct cell-killing, such as, e.g., via the ribotoxic,enzymatic activity of a Shiga toxin effector polypeptide or ribosomebinding and interference with ribosome function due to a non-catalyticmechanism(s).

For certain embodiments of the CD8+ T-cell hyper-immunized,cell-targeting molecules of the present invention, upon contacting acell physically coupled with an extracellular target biomolecule of thecell-targeting binding region, the cell-targeting molecule of theinvention is capable of directly causing the death of the cell, such as,e.g., without the involvement of a untargeted, cytotoxic T-cell (seeSection V-D, supra).

G. Selective Cytotoxicity Among Cell Types

Certain cell-targeting molecules of the present invention have uses inthe selective killing of specific target cells in the presence ofuntargeted, bystander cells. By targeting the delivery of Shiga toxineffector polypeptides of the present invention to specific cells via acell-targeting binding region(s), the cell-targeting molecules of thepresent invention can exhibit cell-type specific, restricted cell-killactivities resulting in the exclusive or preferential killing selectedcell types in the presence of untargeted cells. Similarly, by targetingthe delivery of immunogenic T-cell epitopes to the MHC class I pathwayof target cells, the subsequent presentation of T-cell epitopes andCTL-mediated cytolysis of target cells induced by the cell-targetingmolecules of the invention can be restricted to exclusively orpreferentially killing selected cell types in the presence of untargetedcells. In addition, both the cell-targeted delivery of a cytotoxic,Shiga toxin effector polypeptide region and an immunogenic, T-cellepitope can be accomplished by a single cell-targeting molecule of thepresent invention such that deliver of both potentially cytotoxiccomponents is restricted exclusively or preferentially to target cellsin the presence of untargeted cells.

For certain embodiments, the cell-targeting molecule of the presentinvention is cytotoxic at certain concentrations. In certainembodiments, upon administration of the cell-targeting molecule of thepresent invention to a mixture of cell types, the cytotoxiccell-targeting molecule is capable of selectively killing those cellswhich are physically coupled with an extracellular target biomoleculecompared to cell types not physically coupled with an extracellulartarget biomolecule. For certain embodiments, the cytotoxiccell-targeting molecule of the present invention is capable ofselectively or preferentially causing the death of a specific cell typewithin a mixture of two or more different cell types. This enablestargeting cytotoxic activity to specific cell types with a highpreferentiality, such as a 3-fold cytotoxic effect, over “bystander”cell types that do not express the target biomolecule. Alternatively,the expression of the target biomolecule of the binding region may benon-exclusive to one cell type if the target biomolecule is expressed inlow enough amounts and/or physically coupled in low amounts with celltypes that are not to be targeted. This enables the targetedcell-killing of specific cell types with a high preferentiality, such asa 3-fold cytotoxic effect, over “bystander” cell types that do notexpress significant amounts of the target biomolecule or are notphysically coupled to significant amounts of the target biomolecule.

For certain further embodiments, upon administration of the cytotoxiccell-targeting molecule to two different populations of cell types, thecytotoxic cell-targeting molecule is capable of causing cell death asdefined by the half-maximal cytotoxic concentration (CD₅₀) on apopulation of target cells, whose members express an extracellulartarget biomolecule of the binding region of the cytotoxic cell-targetingmolecule, at a dose at least three-times lower than the CD₅₀ dose of thesame cytotoxic cell-targeting molecule to a population of cells whosemembers do not express an extracellular target biomolecule of thebinding region of the cytotoxic cell-targeting molecule.

For certain embodiments, the cytotoxic activity of a cell-targetingmolecule of the present invention toward populations of cell typesphysically coupled with an extracellular target biomolecule is at least3-fold higher than the cytotoxic activity toward populations of celltypes not physically coupled with any extracellular target biomoleculeof the binding region. According to the present invention, selectivecytotoxicity may be quantified in terms of the ratio (a/b) of (a)cytotoxicity towards a population of cells of a specific cell typephysically coupled with a target biomolecule of the binding region to(b) cytotoxicity towards a population of cells of a cell type notphysically coupled with a target biomolecule of the binding region. Incertain embodiments, the cytotoxicity ratio is indicative of selectivecytotoxicity which is at least 3-fold, 5-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold,250-fold, 500-fold, 750-fold, or 1000-fold higher for populations ofcells or cell types physically coupled with a target biomolecule of thebinding region compared to populations of cells or cell types notphysically coupled with a target biomolecule of the binding region.

For certain embodiments, the preferential cell-killing function orselective cytotoxicity of a cell-targeting molecule of the presentinvention is due to an additional exogenous material (e.g. a cytotoxicmaterial) and/or heterologous, T-cell epitope present in a Shiga toxineffector polypeptide of the present invention and not necessarily aresult of the catalytic activity of a Shiga toxin effector polypeptideregion.

This preferential cell-killing function allows a targeted cell to bekilled by certain cytotoxic, cell-targeting molecules of the presentinvention under varied conditions and in the presence of non-targetedbystander cells, such as ex vivo manipulated mixtures of cell types, invitro cultured tissues with mixtures of cell types, or in vivo in thepresence of multiple cell types (e.g. in situ or in a native locationwithin a multicellular organism).

H. Delivery of Additional Exogenous Material into the Interior ofTargeted Cells

In addition to cytotoxic, cytostatic, and immune stimulationapplications, cell-targeting molecules of the present inventionoptionally may be used for targeted intracellular delivery functions,such as, e.g., in applications involving information gathering anddiagnostic functions.

Because the cell-targeting molecules of the invention, including reducedcytotoxicity and/or nontoxic forms thereof, are capable of enteringcells physically coupled with an extracellular target biomoleculerecognized by the cell-targeting molecule's binding region, certainembodiments of the cell-targeting molecules of the invention may be usedto deliver additional exogenous materials into the interior of targetedcell types. For example, non-toxic variants of the cytotoxic,cell-targeting molecules of the invention, or optionally cytotoxicvariants, may be used to deliver additional exogenous materials toand/or label the interiors of cells physically coupled with anextracellular target biomolecule of the binding region of thecell-targeting molecule. Various types of cells and/or cell populationswhich express target biomolecules to at least one cellular surface maybe targeted by the cell-targeting molecules of the invention forreceiving exogenous materials. The functional components of the presentinvention are modular, in that various Shiga toxin effectorpolypeptides, additional exogenous materials, and binding regions may beassociated with each other to provide cell-targeting molecules suitablefor diverse applications involving cargo delivery, such as, e.g.,non-invasive, in vivo imaging of tumor cells.

This delivery of exogenous material function of certain cell-targetingmolecules of the present invention may be accomplished under variedconditions and in the presence of non-targeted bystander cells, such as,e.g., an ex vivo manipulated target cell, a target cell cultured invitro, a target cell within a tissue sample cultured in vitro, or atarget cell in an in vivo setting like within a multicellular organism.Furthermore, the selective delivery of exogenous material to certaincells by certain cell-targeting molecules of the present invention maybe accomplished under varied conditions and in the presence ofnon-targeted bystander cells, such as ex vivo manipulated mixtures ofcell types, in vitro cultured tissues with mixtures of cell types, or invivo in the presence of multiple cell types (e.g. in situ or in a nativelocation within a multicellular organism).

Shiga toxin effector polypeptides and cell-targeting molecules which arenot capable, such as a certain concentration ranges, of killing a targetcell and/or delivering an embedded or inserted epitope for cell-surfacepresentation by a MHC molecule of a target cell may still be useful fordelivering exogenous materials into cells, such as, e.g., detectionpromoting agents.

For certain embodiments, the Shiga toxin effector polypeptides of thepresent invention exhibits low to zero cytotoxicity and thus arereferred to herein as “noncytotoxic and/or reduced cytotoxic.” Forcertain embodiments, the cell-targeting molecule of the presentinvention exhibits low to zero cytotoxicity and may be referred to as“noncytotoxic” and/or “reduced cytotoxic variants.” For example, certainembodiments of the molecules of the present invention do not exhibit asignificant level of Shiga toxin based cytotoxicity wherein at doses ofless than 1000 nM, 500 nM, 100 nM, 75 nM, 50 nM, there is no significantamount of cell death as compared to the appropriate reference molecule,such as, e.g., as measured by an assay known to the skilled workerand/or described herein. For certain further embodiments, the moleculesof the present invention do not exhibit any toxicity at dosages of 1-100pg per kg of a mammalian recipient. Reduced-cytotoxic variants may stillbe cytotoxic at certain concentrations or dosages but exhibit reducedcytotoxicity, such as, e.g., are not capable of exhibiting a significantlevel of Shiga toxin cytotoxicity in certain situations.

Shiga toxin effector polypeptides of the present invention, and certaincell-targeting molecules comprising the same, can be renderednon-cytotoxic, such as, e.g., via the addition of one or more amino acidsubstitutions known to the skilled worker to inactive a Shiga toxin ASubunit and/or Shiga toxin effector polypeptide, including exemplarysubstitutions described herein. The non-cytotoxic and reduced cytotoxicvariants of the cell-targeting molecules of the present invention may bein certain situations more suitable for delivery of additional exogenousmaterials than more cytotoxic variants.

Information Gathering for Diagnostic Functions

In certain cell-targeting molecules of the present invention have usesin the in vitro and/or in vivo detection of specific cells, cell types,and/or cell populations, as well as specific subcellular compartments ofany of the aforementioned. Reduced-cytotoxicity and/or nontoxic forms ofthe cytotoxic, cell-targeting molecules of the invention that areconjugated to detection promoting agents optionally may be used fordiagnostic functions, such as for companion diagnostics used inconjunction with a therapeutic regimen comprising the same or a relatedbinding region, such as, e.g., a binding region with high-affinitybinding to the same target biomolecule, an overlapping epitope, and/orthe same epitope.

In certain embodiments, the cell-targeting molecules described hereinare used for both diagnosis and treatment, or for diagnosis alone. Whenthe same cytotoxic cell-targeting molecule is used for both diagnosisand treatment, for certain embodiments of the present invention thecell-targeting molecule variant which incorporates a detection promotingagent for diagnosis may have its cytotoxicity reduced or may be renderednontoxic by catalytic inactivation of its Shiga toxin effectorpolypeptide region(s) via one or more amino acid substitutions,including exemplary substitutions described herein. For example, certainnontoxic variants of the cell-targeting molecules of the presentinvention exhibit less than 5%, 4%, 3%, 2%, or 1% death of target cellsafter administration of a dose less than 1 mg/kg. Reduced-cytotoxicityvariants may still be cytotoxic at certain concentrations or dosages butexhibit reduced cytotoxicity, such as, e.g., are not capable ofexhibiting a significant level of Shiga toxin cytotoxicity as describedherein.

The ability to conjugate detection promoting agents known in the art tovarious cell-targeting molecules of the present invention providesuseful compositions for the detection of certain cells, such as, e.g.,cancer, tumor, immune, and/or infected cells. These diagnosticembodiments of the cell-targeting molecules of the invention may be usedfor information gathering via various imaging techniques and assaysknown in the art. For example, diagnostic embodiments of thecell-targeting molecules of the invention may be used for informationgathering via imaging of intracellular organelles (e.g. endocytotic,Golgi, endoplasmic reticulum, and cytosolic compartments) of individualcancer cells, immune cells, and/or infected cells in a patient or biopsysample.

Various types of information may be gathered using the diagnosticembodiments of the cell-targeting molecules of the invention whether fordiagnostic uses or other uses. This information may be useful, forexample, in diagnosing neoplastic cell types, determining therapeuticsusceptibilities of a patient's disease, assaying the progression ofanti-neoplastic therapies over time, assaying the progression ofimmunomodulatory therapies over time, assaying the progression ofantimicrobial therapies over time, evaluating the presence of infectedcells in transplantation materials, evaluating the presence of unwantedcell types in transplantation materials, and/or evaluating the presenceof residual tumor cells after surgical excision of a tumor mass.

For example, subpopulations of patients might be ascertained usinginformation gathered using the diagnostic variants of the cell-targetingmolecules of the invention, and then individual patients could befurther categorized into subpopulations based on their uniquecharacteristic(s) revealed using those diagnostic embodiments. Forexample, the effectiveness of specific pharmaceuticals or therapiesmight be a criterion used to define a patient subpopulation. Forexample, a nontoxic diagnostic variant of a particular cytotoxic,cell-targeting molecule of the invention may be used to differentiatewhich patients are in a class or subpopulation of patients predicted torespond positively to a cytotoxic variant of that cell-targetingmolecule of the invention. Accordingly, associated methods for patientidentification, patient stratification, and diagnosis usingcell-targeting molecules of the present invention, including non-toxicvariants of cytotoxic, cell-targeting molecules of the presentinvention, are considered to be within the scope of the presentinvention.

The expression of the target biomolecule by a cell need not be native inorder for cell-targeting by a cell-targeting molecule of the presentinvention, such as, e.g., for direct cell-kill, indirect cell-kill,delivery of exogenous materials like T-cell epitopes, and/or informationgathering. Cell surface expression of the target biomolecule could bethe result of an infection, the presence of a pathogen, and/or thepresence of an intracellular microbial pathogen. Expression of a targetbiomolecule could be artificial such as, for example, by forced orinduced expression after infection with a viral expression vector, seee.g. adenoviral, adeno-associated viral, and retroviral systems. Anexample of inducing expression of a target biomolecule is theupregulation of CD38 expression of cells exposed to retinoids, likeall-trans retinoic acid and various synthetic retinoids, or any retinoicacid receptor (RAR) agonist (Drach J et al., Cancer Res 54: 1746-52(1994); Uruno A et al., J Leukoc Biol 90: 235-47 (2011)). Expression ofCD30 can be induced in both B-cells and T-cells by exposure to bymitogens, phytohemagglutinin (PHA), staphylococcal protein A, EBV virus,human T-cell leukemia virus 1 or 2 (HTLV-1 or HTLV-2) (see e.g. Stein Het al., Blood 66: 848-58 (1985)). In another example, CD20, HER2, andEGFR expression may be induced by exposing a cell to ionizing radiation(Wattenberg M et al., Br J Cancer 110: 1472-80 (2014)). Further, PSMAexpression is upregulated in response to androgen deprivation (see e.g.Chang S et al., Cancer 88: 407-15 (2000); Meller B et al., EJNMMI Res 5:66 (2015)).

VI. Production, Manufacture, and Purification of Shiga Toxin EffectorPolypeptides of the Invention and Cell-Targeting Molecules Comprisingthe Same

The Shiga toxin effector polypeptides and certain cell-targetingmolecules of the present invention may be produced using techniques wellknown to those of skill in the art. For example, Shiga toxin effectorpolypeptides and cell-targeting molecules of the invention may bemanufactured by standard synthetic methods, by use of recombinantexpression systems, or by any other suitable method. Thus, Shiga toxineffector polypeptides and cell-targeting molecules of the invention maybe synthesized in a number of ways, including, e.g. methods comprising:(1) synthesizing a polypeptide or polypeptide component of acell-targeting molecule using standard solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolating andpurifying the final polypeptide compound product; (2) expressing apolynucleotide that encodes a protein or protein component of acell-targeting molecule of the invention in a host cell and recoveringthe expression product from the host cell or host cell culture; or (3)cell-free, in vitro expression of a polynucleotide encoding apolypeptide or polypeptide component of a cell-targeting molecule of theinvention, and recovering the expression product; or by any combinationof the methods of (1), (2) or (3) to obtain fragments of the proteincomponent, subsequently joining (e.g. ligating) the peptide orpolypeptide fragments to obtain a polypeptide component, and recoveringthe polypeptide component.

It may be preferable to synthesize a Shiga toxin effector polypeptide ofthe present invention, cell-targeting molecule of the present invention,or a protein component of a cell-targeting molecule of the invention bymeans of solid-phase or liquid-phase peptide synthesis. Polypeptides andcell-targeting molecules of the present invention may suitably bemanufactured by standard synthetic methods. Thus, peptides may besynthesized by, e.g. methods comprising synthesizing the peptide bystandard solid-phase or liquid-phase methodology, either stepwise or byfragment assembly, and isolating and purifying the final peptideproduct. In this context, reference may be made to WO 1998/011125 or,inter alia, Fields G et al., Principles and Practice of Solid-PhasePeptide Synthesis (Synthetic Peptides, Grant G, ed., Oxford UniversityPress, U.K., 2nd ed., 2002) and the synthesis examples therein.

Shiga toxin effector polypeptides and cell-targeting molecules of thepresent invention may be prepared (produced and purified) usingrecombinant techniques well known in the art. In general, methods forpreparing proteins by culturing host cells transformed or transfectedwith a vector comprising the encoding polynucleotide and purifying orrecovering the protein from cell culture are described in, e.g.,Sambrook J et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, NY, U.S., 1989); Dieffenbach C et al., PCRPrimer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, N.Y.,U.S., 1995). Any suitable host cell may be used to produce a polypeptideand/or cell-targeting protein of the invention. Host cells may be cellsstably or transiently transfected, transformed, transduced or infectedwith one or more expression vectors which drive expression of apolypeptide of the invention. In addition, a Shiga toxin effectorpolypeptide and/or cell-targeting molecule of the invention may beproduced by modifying the polynucleotide encoding a polypeptide orcell-targeting protein of the invention that result in altering one ormore amino acids or deleting or inserting one or more amino acids inorder to achieve desired properties, such as changed cytotoxicity,changed cytostatic effects, and/or changed serum half-life.

There are a wide variety of expression systems which may be chosen toproduce a polypeptide or cell-targeting protein of the presentinvention. For example, host organisms for expression of cell-targetingproteins of the invention include prokaryotes, such as E. coli and B.subtilis, eukaryotic cells, such as yeast and filamentous fungi (like S.cerevisiae, P. pastoris, A. awamori, and K. lactis), algae (like C.reinhardtii), insect cell lines, mammalian cells (like CHO cells), plantcell lines, and eukaryotic organisms such as transgenic plants (like A.thaliana and N. benthamiana).

Accordingly, the present invention also provides methods for producing aShiga toxin effector polypeptide and/or cell-targeting molecule of thepresent invention according to above recited methods and using apolynucleotide encoding part or all of a polypeptide of the invention ora protein component of a cell-targeting protein of the invention, anexpression vector comprising at least one polynucleotide of theinvention capable of encoding part or all of a polypeptide orcell-targeting protein of the invention when introduced into a hostcell, and/or a host cell comprising a polynucleotide or expressionvector of the invention.

When a protein is expressed using recombinant techniques in a host cellor cell-free system, it is advantageous to separate (or purify) thedesired protein away from other components, such as host cell factors,in order to obtain preparations that are of high purity or aresubstantially homogeneous. Purification can be accomplished by methodswell known in the art, such as centrifugation techniques, extractiontechniques, chromatographic and fractionation techniques (e.g. sizeseparation by gel filtration, charge separation by ion-exchange column,hydrophobic interaction chromatography, reverse phase chromatography,chromatography on silica or cation-exchange resins such as DEAE and thelike, chromatofocusing, and Protein A Sepharose chromatography to removecontaminants), and precipitation techniques (e.g. ethanol precipitationor ammonium sulfate precipitation). Any number of biochemicalpurification techniques may be used to increase the purity of apolypeptide and/or cell-targeting molecule of the present invention. Incertain embodiments, the polypeptides and cell-targeting molecules ofthe invention may optionally be purified in homo-multimeric forms (e.g.a molecular complex comprising two or more polypeptides orcell-targeting molecules of the invention).

In the Examples below are descriptions of non-limiting examples ofmethods for producing exemplary, Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, as well as specificbut non-limiting aspects of production methods.

VII. Pharmaceutical and Diagnostic Compositions ComprisingCell-Targeting Molecules of the Present Invention

The present invention provides Shiga toxin effector polypeptides andcell-targeting molecules for use, alone or in combination with one ormore additional therapeutic agents, in a pharmaceutical composition, fortreatment or prophylaxis of conditions, diseases, disorders, or symptomsdescribed in further detail below (e.g. cancers, malignant tumors,non-malignant tumors, growth abnormalities, immune disorders, andmicrobial infections). The present invention further providespharmaceutical compositions comprising a Shiga toxin polypeptide orcell-targeting molecule of the present invention, or a pharmaceuticallyacceptable salt or solvate thereof, according to the invention, togetherwith at least one pharmaceutically acceptable carrier, excipient, orvehicle. In certain embodiments, the pharmaceutical composition of thepresent invention may comprise homo-multimeric and/or hetero-multimericforms of a Shiga toxin effector polypeptides or cell-targeting moleculeof the present invention. The pharmaceutical compositions of theinvention are useful in methods of treating, ameliorating, or preventinga disease, condition, disorder, or symptom described in further detailbelow. Each such disease, condition, disorder, or symptom is envisionedto be a separate embodiment with respect to uses of a pharmaceuticalcomposition according to the invention. The invention further providespharmaceutical compositions for use in at least one method of treatmentaccording to the invention, as described in more detail below.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to any organism, commonly vertebrates such ashumans and animals, which presents symptoms, signs, and/or indicationsof at least one disease, disorder, or condition. These terms includemammals such as the non-limiting examples of primates, livestock animals(e.g. cattle, horses, pigs, sheep, goats, etc.), companion animals (e.g.cats, dogs, etc.) and laboratory animals (e.g. mice, rabbits, rats,etc.).

As used herein, “treat,” “treating,” or “treatment” and grammaticalvariants thereof refer to an approach for obtaining beneficial ordesired clinical results. The terms may refer to slowing the onset orrate of development of a condition, disorder or disease, reducing oralleviating symptoms associated with it, generating a complete orpartial regression of the condition, or some combination of any of theabove. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, reduction oralleviation of symptoms, diminishment of extent of disease,stabilization (e.g. not worsening) of state of disease, delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treat,” “treating,” or “treatment” can also meanprolonging survival relative to expected survival time if not receivingtreatment. A subject (e.g. a human) in need of treatment may thus be asubject already afflicted with the disease or disorder in question. Theterms “treat,” “treating,” or “treatment” includes inhibition orreduction of an increase in severity of a pathological state or symptomsrelative to the absence of treatment, and is not necessarily meant toimply complete cessation of the relevant disease, disorder, orcondition. With regard to tumors and/or cancers, treatment includesreduction in overall tumor burden and/or individual tumor size.

As used herein, the terms “prevent,” “preventing,” “prevention” andgrammatical variants thereof refer to an approach for preventing thedevelopment of, or altering the pathology of, a condition, disease, ordisorder. Accordingly, “prevention” may refer to prophylactic orpreventive measures. For the purposes of this invention, beneficial ordesired clinical results include, but are not limited to, prevention orslowing of symptoms, progression or development of a disease, whetherdetectable or undetectable. A subject (e.g. a human) in need ofprevention may thus be a subject not yet afflicted with the disease ordisorder in question. The term “prevention” includes slowing the onsetof disease relative to the absence of treatment, and is not necessarilymeant to imply permanent prevention of the relevant disease, disorder orcondition. Thus “preventing” or “prevention” of a condition may incertain contexts refer to reducing the risk of developing the condition,or preventing or delaying the development of symptoms associated withthe condition.

As used herein, an “effective amount” or “therapeutically effectiveamount” is an amount or dose of a composition (e.g. a therapeuticcomposition, compound, or agent) that produces at least one desiredtherapeutic effect in a subject, such as preventing or treating a targetcondition or beneficially alleviating a symptom associated with thecondition. The most desirable therapeutically effective amount is anamount that will produce a desired efficacy of a particular treatmentselected by one of skill in the art for a given subject in need thereof.This amount will vary depending upon a variety of factors understood bythe skilled worker, including but not limited to the characteristics ofthe therapeutic composition (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type, disease stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a composition and adjusting the dosage accordingly(see e.g. Remington: The Science and Practice of Pharmacy (Gennaro A,ed., Mack Publishing Co., Easton, Pa., U.S., 19th ed., 1995)).

Diagnostic compositions of the present invention comprise acell-targeting molecule of the present invention and one or moredetection promoting agents.

When producing or manufacturing a diagnostic composition of the presentinvention, a cell-targeting molecule of the present invention may bedirectly or indirectly linked to one or more detection promoting agents.There are numerous standard techniques known to the skilled worker forincorporating, affixing, and/or conjugating various detection promotingagents to proteins or proteinaceous components of molecules, especiallyto immunoglobulins and immunoglobulin-derived domains.

There are numerous detection promoting agents known to the skilledworker, such as isotopes, dyes, colorimetric agents, contrast enhancingagents, fluorescent agents, bioluminescent agents, and magnetic agents,which can be operably linked to the polypeptides or cell-targetingmolecules of the invention for information gathering methods, such asfor diagnostic and/or prognostic applications to diseases, disorders, orconditions of an organism (see e.g. Cai W et al., J Nucl Med 48: 304-10(2007); Nayak T, Brechbiel M, Bioconjug Chem 20: 825-41 (2009); PaudyalP et al., Oncol Rep 22: 115-9 (2009); Qiao J et al., PLoS ONE 6: e18103(2011); Sano K et al., Breast Cancer Res 14: R61 (2012)). These agentsmay be associated with, linked to, and/or incorporated within thepolypeptide or cell-targeting molecule of the invention at any suitableposition. For example, the linkage or incorporation of the detectionpromoting agent may be via an amino acid residue(s) of a molecule of thepresent invention or via some type of linkage known in the art,including via linkers and/or chelators. The incorporation of the agentis in such a way to enable the detection of the presence of thediagnostic composition in a screen, assay, diagnostic procedure, and/orimaging technique.

Similarly, there are numerous imaging approaches known to the skilledworker, such as non-invasive in vivo imaging techniques commonly used inthe medical arena, for example: computed tomography imaging (CTscanning), optical imaging (including direct, fluorescent, andbioluminescent imaging), magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT), ultrasound, and x-ray computed tomography imaging.

VIII. Production or Manufacture of Pharmaceutical and/or DiagnosticCompositions Comprising Cell-Targeting Molecules of the PresentInvention

Pharmaceutically acceptable salts or solvates of any of the Shiga toxineffector polypeptides and cell-targeting molecules of the presentinvention are within the scope of the present invention.

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, aproteinaceous compound or pharmaceutically acceptable salt thereofaccording to the invention) and a solvent. The solvent in thisconnection may, for example, be water, ethanol or anotherpharmaceutically acceptable, typically small-molecular organic species,such as, but not limited to, acetic acid or lactic acid. When thesolvent in question is water, such a solvate is normally referred to asa hydrate.

Polypeptides and proteins of the present invention, or salts thereof,may be formulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a molecule of the present invention, or a salt thereof, in apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” includes any of the standard pharmaceuticalcarriers. Pharmaceutically acceptable carriers for therapeutic moleculeuse are well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences (Mack Publishing Co. (A.Gennaro, ed., 1985). As used herein, “pharmaceutically acceptablecarrier” includes any and all physiologically acceptable, i.e.compatible, solvents, dispersion media, coatings, antimicrobial agents,isotonic, and absorption delaying agents, and the like. Pharmaceuticallyacceptable carriers or diluents include those used in formulationssuitable for oral, rectal, nasal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. Exemplary pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Examples of suitable aqueous and nonaqueous carriers thatmay be employed in the pharmaceutical compositions of the inventioninclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyloleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. In certain embodiments, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Depending onselected route of administration, the protein or other pharmaceuticalcomponent may be coated in a material intended to protect the compoundfrom the action of low pH and other natural inactivating conditions towhich the active protein may encounter when administered to a patient bya particular route of administration.

The formulations of the pharmaceutical compositions of the invention mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. In such form, thecomposition is divided into unit doses containing appropriate quantitiesof the active component. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Subcutaneous or transdermalmodes of administration may be particularly suitable for therapeuticproteins described herein.

The pharmaceutical compositions of the present invention may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Preventing the presence of microorganismsmay be ensured both by sterilization procedures, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, suchas sugars, sodium chloride, and the like into the compositions, may alsobe desirable. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as, aluminum monostearate and gelatin.

A pharmaceutical composition of the present invention also optionallyincludes a pharmaceutically acceptable antioxidant. Exemplarypharmaceutically acceptable antioxidants are water soluble antioxidantssuch as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; oil-soluble antioxidants,such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and thelike; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, andthe like.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or a combination of different polypeptidesand/or cell-targeting molecules of the invention, or an ester, salt oramide of any of the foregoing, and at least one pharmaceuticallyacceptable carrier.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g., sugars andpolyalcohols such as mannitol, sorbitol, or sodium chloride, may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating apolypeptide or cell-targeting molecule of the invention in the requiredamount in an appropriate solvent with one or a combination ofingredients described above, as required, followed by sterilizationmicrofiltration. Dispersions may be prepared by incorporating the activecompound into a sterile vehicle that contains a dispersion medium andother ingredients, such as those described above. In the case of sterilepowders for the preparation of sterile injectable solutions, the methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient in addition to any additionaldesired ingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a polypeptide and/orcell-targeting molecule of the invention is designed to be administeredby, e.g. intravenous, cutaneous or subcutaneous injection, the bindingagent will be in the form of a pyrogen-free, parenterally acceptableaqueous solution. Methods for preparing parenterally acceptable proteinsolutions, taking into consideration appropriate pH, isotonicity,stability, and the like, are within the skill in the art. A preferredpharmaceutical composition for intravenous, cutaneous, or subcutaneousinjection will contain, in addition to binding agents, an isotonicvehicle such as sodium chloride injection, Ringer's injection, dextroseinjection, dextrose and sodium chloride injection, lactated Ringer'sinjection, or other vehicle as known in the art. A pharmaceuticalcomposition of the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives well known tothose of skill in the art.

As described elsewhere herein, a polypeptide and/or cell-targetingmolecule of the present invention may be prepared with carriers thatwill protect the active therapeutic agent against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art (see e.g.Sustained and Controlled Release Drug Delivery Systems (Robinson J, ed.,Marcel Dekker, Inc., NY, U.S., 1978)).

In certain embodiments, the composition of the present invention (e.g. apharmaceutical and/or diagnostic composition) may be formulated toensure a desired in vivo distribution of a cell-targeting molecule ofthe present invention. For example, the blood-brain barrier excludesmany large and/or hydrophilic compounds. To target a therapeuticmolecule or composition of the present invention to a particular in vivolocation, they can be formulated, for example, in liposomes which maycomprise one or more moieties that are selectively transported intospecific cells or organs, thus enhancing targeted drug delivery.Exemplary targeting moieties include folate or biotin; mannosides;antibodies; surfactant protein A receptor; p120 catenin and the like.

Pharmaceutical compositions include parenteral formulations designed tobe used as implants or particulate systems. Examples of implants aredepot formulations composed of polymeric or hydrophobic components suchas emulsions, ion exchange resins, and soluble salt solutions. Examplesof particulate systems are microspheres, microparticles, nanocapsules,nanospheres, and nanoparticles (see e.g. Honda M et al., Int JNanomedicine 8: 495-503 (2013); Sharma A et al., Biomed Res Int 2013:960821 (2013); Ramishetti S, Huang L, Ther Deliv 3: 1429-45 (2012)).Controlled release formulations may be prepared using polymers sensitiveto ions, such as, e.g. liposomes, polaxamer 407, and hydroxyapatite.

IX. Polynucleotides, Expression Vectors, and Host Cells of the PresentInvention

Beyond the polypeptides and cell-targeting molecules of the presentinvention, the polynucleotides that encode the polypeptides andcell-targeting molecules of the invention, or functional portionsthereof, are also encompassed within the scope of the present invention.The term “polynucleotide” is equivalent to the term “nucleic acid,” eachof which includes one or more of: polymers of deoxyribonucleic acids(DNAs), polymers of ribonucleic acids (RNAs), analogs of these DNAs orRNAs generated using nucleotide analogs, and derivatives, fragments andhomologs thereof. The polynucleotide of the present invention may besingle-, double-, or triple-stranded. Such polynucleotides arespecifically disclosed to include all polynucleotides capable ofencoding an exemplary protein, for example, taking into account thewobble known to be tolerated in the third position of RNA codons, yetencoding for the same amino acid as a different RNA codon (see StothardP, Biotechniques 28: 1102-4 (2000)).

In one aspect, the present invention provides polynucleotides whichencode a Shiga toxin effector polypeptide and/or cell-targeting moleculeof the present invention, or a fragment or derivative thereof. Thepolynucleotides may include, e.g., a nucleic acid sequence encoding apolypeptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or more, identical to a polypeptide comprising one of the amino acidsequences of a polypeptide or cell-targeting molecule of the presentinvention. The invention also includes polynucleotides comprisingnucleotide sequences that hybridize under stringent conditions to apolynucleotide which encodes Shiga toxin effector polypeptide and/orcell-targeting molecule of the invention, or a fragment or derivativethereof, or the antisense or complement of any such sequence.

Derivatives or analogs of the molecules of the present invention (e.g.,Shiga toxin effector polypeptides of the present invention andcell-targeting molecules comprising the same) include, inter alia,polynucleotide (or polypeptide) molecules having regions that aresubstantially homologous to the polynucleotides (or Shiga toxin effectorpolypeptides and cell-targeting molecules of the present invention),e.g. by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99%identity (with a preferred identity of 80-99%) over a polynucleotide (orpolypeptide) sequence of the same size or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art. An exemplary program is the GAP program (WisconsinSequence Analysis Package, Version 8 for UNIX, Genetics Computer Group,University Research Park, Madison, Wis., U.S.) using the defaultsettings, which uses the algorithm of Smith T, Waterman M, Adv Appl Math2: 482-9 (1981). Also included are polynucleotides capable ofhybridizing to the complement of a sequence encoding the cell-targetingproteins of the invention under stringent conditions (see e.g. Ausubel Fet al., Current Protocols in Molecular Biology (John Wiley & Sons, NewYork, N.Y., U.S., 1993)), and below. Stringent conditions are known tothose skilled in the art and may be found, e.g., in Current Protocols inMolecular Biology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6(1989)).

The present invention further provides expression vectors that comprisethe polynucleotides within the scope of the present invention. Thepolynucleotides capable of encoding the Shiga toxin effectorpolypeptides and/or cell-targeting molecules of the invention may beinserted into known vectors, including bacterial plasmids, viral vectorsand phage vectors, using material and methods well known in the art toproduce expression vectors. Such expression vectors will include thepolynucleotides necessary to support production of contemplated Shigatoxin effector polypeptides and/or cell-targeting molecules of theinvention within any host cell of choice or cell-free expression systems(e.g. pTxb1 and pIVEX2.3). The specific polynucleotides comprisingexpression vectors for use with specific types of host cells orcell-free expression systems are well known to one of ordinary skill inthe art, can be determined using routine experimentation, and/or may bepurchased.

The term “expression vector,” as used herein, refers to apolynucleotide, linear or circular, comprising one or more expressionunits. The term “expression unit” denotes a polynucleotide segmentencoding a polypeptide of interest and capable of providing expressionof the nucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. An expression vector contains one or more expressionunits. Thus, in the context of the present invention, an expressionvector encoding a Shiga toxin effector polypeptide and/or cell-targetingmolecule of the invention comprising a single polypeptide chain includesat least an expression unit for the single polypeptide chain, whereas aprotein comprising, e.g. two or more polypeptide chains (e.g. one chaincomprising a V_(L) domain and a second chain comprising a V_(H) domainlinked to a toxin effector polypeptide) includes at least two expressionunits, one for each of the two polypeptide chains of the protein. Forexpression of multi-chain cell-targeting proteins of the invention, anexpression unit for each polypeptide chain may also be separatelycontained on different expression vectors (e.g. expression may beachieved with a single host cell into which expression vectors for eachpolypeptide chain has been introduced).

Expression vectors capable of directing transient or stable expressionof polypeptides and proteins are well known in the art. The expressionvectors generally include, but are not limited to, one or more of thefollowing: a heterologous signal sequence or peptide, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, each of which is well known inthe art. Optional regulatory control sequences, integration sequences,and useful markers that can be employed are known in the art.

The term “host cell” refers to a cell which can support the replicationor expression of the expression vector. Host cells may be prokaryoticcells, such as E. coli or eukaryotic cells (e.g. yeast, insect,amphibian, bird, or mammalian cells). Creation and isolation of hostcell lines comprising a polynucleotide of the invention or capable ofproducing a polypeptide and/or cell-targeting molecule of the presentinvention can be accomplished using standard techniques known in theart.

Shiga toxin effector polypeptides and/or proteins within the scope ofthe present invention may be variants or derivatives of the polypeptidesand molecules described herein that are produced by modifying thepolynucleotide encoding a polypeptide and/or proteinaceous component ofa cell-targeting molecule by altering one or more amino acids ordeleting or inserting one or more amino acids that may render it moresuitable to achieve desired properties, such as more optimal expressionby a host cell.

X. Molecules of the Present Invention Immobilized on Solid Substrates

Certain embodiments of the present invention include a molecule of thepresent invention (e.g. a Shiga toxin effector polypeptide, acell-targeting molecule, fusion protein, or polynucleotide of thepresent invention), or any effector fragment thereof, immobilized on asolid substrate. Solid substrates contemplated herein include, but arenot limited to, microbeads, nanoparticles, polymers, matrix materials,microarrays, microtiter plates, or any solid surface known in the art(see e.g. U.S. Pat. No. 7,771,955). In accordance with theseembodiments, a molecule of the present invention may be covalently ornon-covalently linked to a solid substrate, such as, e.g., a bead,particle, or plate, using techniques known to the skilled worker (seee.g. Jung Y et al., Analyst 133: 697-701 (2008)). Immobilized moleculesof the invention may be used for screening applications using techniquesknown in the art (see e.g. Bradbury A et al., Nat Biotechnol 29: 245-54(2011); Sutton C, Br J Pharmacol 166: 457-75 (2012); Diamante L et al.,Protein Eng Des Sel 26: 713-24 (2013); Houlihan G et al., J ImmunolMethods 405: 47-56 (2014)).

Non-limiting examples of solid substrates to which a molecule of theinvention may be immobilized on include: microbeads, nanoparticles,polymers, nanopolymers, nanotubes, magnetic beads, paramagnetic beads,superparamagnetic beads, streptavidin coated beads, reverse-phasemagnetic beads, carboxy terminated beads, hydrazine terminated beads,silica (sodium silica) beads and iminodiacetic acid (IDA)-modifiedbeads, aldehyde-modified beads, epoxy-activated beads,diaminodipropylamine (DADPA)-modified beads (beads with primary aminesurface group), biodegradable polymeric beads, polystyrene substrates,amino-polystyrene particles, carboxyl-polystyrene particles,epoxy-polystyrene particles, dimethylamino-polystyrene particles,hydroxy-polystyrene particles, colored particles, flow cytometryparticles, sulfonate-polystyrene particles, nitrocellulose surfaces,reinforced nitrocellulose membranes, nylon membranes, glass surfaces,activated glass surfaces, activated quartz surfaces, polyvinylidenedifluoride (PVDF) membranes, polyacrylamide-based substrates, poly-vinylchloride substrates, poly-methyl methacrylate substrates, poly(dimethylsiloxane) substrates, and photopolymers which contain photoreactivespecies (such as nitrenes, carbenes, and ketyl radicals) capable offorming covalent linkages. Other examples of solid substrates to which amolecule of the invention may be immobilized on are commonly used inmolecular display systems, such as, e.g., cellular surfaces, phages, andvirus particles.

XI. Delivery Devices and Kits

In certain embodiments, the invention relates to a device comprising oneor more compositions of matter of the present invention, such as apharmaceutical composition or diagnostic composition, for delivery to asubject in need thereof. Thus, a delivery device comprising one or morecompositions of the present invention can be used to administer to apatient a composition of matter of the present invention by variousdelivery methods, including: intravenous, subcutaneous, intramuscular orintraperitoneal injection; oral administration; transdermaladministration; pulmonary or transmucosal administration; administrationby implant, osmotic pump, cartridge or micro pump; or by other meansrecognized by a person of skill in the art.

Also within the scope of the present invention are kits comprising atleast one composition of matter of the invention, and optionally,packaging and instructions for use. Kits may be useful for drugadministration and/or diagnostic information gathering. A kit of theinvention may optionally comprise at least one additional reagent (e.g.,standards, markers and the like). Kits typically include a labelindicating the intended use of the contents of the kit. The kit mayfurther comprise reagents and other tools for detecting a cell type(e.g. a tumor cell) in a sample or in a subject, or for diagnosingwhether a patient belongs to a group that responds to a therapeuticstrategy which makes use of a compound, composition, or related methodof the present invention, e.g., such as a method described herein.

XII. Methods for Using Cell-Targeting Molecules of the Present Inventionand/or Pharmaceutical and/or Diagnostic Compositions Thereof

Generally, it is an object of the present invention to providepharmacologically active agents, as well as compositions comprising thesame, that can be used in the prevention and/or treatment of diseases,disorders, and conditions, such as certain cancers, tumors, growthabnormalities, immune disorders, or further pathological conditionsmentioned herein. Accordingly, the present invention provides methods ofusing the polypeptides, cell-targeting molecules, and pharmaceuticalcompositions of the invention for the targeted killing of cells, fordelivering additional exogenous materials into targeted cells, forlabeling of the interiors of targeted cells, for collecting diagnosticinformation, for the delivering of T-cell epitopes to the MHC class Ipresentation pathway of target cells, and for treating diseases,disorders, and conditions as described herein. For example, the methodsof the present invention may be used to prevent or treat cancers, cancerinitiation, tumor initiation, metastasis, and/or disease reoccurrence.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions, and/or methods that havecertain advantages compared to the agents, compositions, and/or methodsthat are currently known in the art. Accordingly, the present inventionprovides methods of using Shiga toxin effector polypeptides andcell-targeting molecules with specified protein sequences andpharmaceutical compositions thereof. For example, any of the amino acidsequences in SEQ ID NOs: 6-354 and 370-513 may be specifically utilizedas a component of the cell-targeting molecule used in the followingmethods or any method for using a cell-targeting molecule known to theskilled worker, such as, e.g., various methods described in WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, WO 2015/191764, US20150259428, 62/168,758,62/168,759, 62/168,760, 62/168,761, 62/168,762, 62/168,763, andPCT/US2016/016580.

The present invention provides methods of killing a cell comprising thestep of contacting the cell, either in vitro or in vivo, with a Shigatoxin effector polypeptide, cell-targeting molecule, or pharmaceuticalcomposition of the present invention. The Shiga toxin effectorpolypeptides, cell-targeting molecules, and pharmaceutical compositionsof the present invention can be used to kill a specific cell type uponcontacting a cell or cells with one of the claimed compositions ofmatter. In certain embodiments, a cell-targeting molecule orpharmaceutical composition of the present invention can be used to killspecific cell types in a mixture of different cell types, such asmixtures comprising cancer cells, infected cells, and/or hematologicalcells. In certain embodiments, a cell-targeting molecule, orpharmaceutical composition of the present invention can be used to killcancer cells in a mixture of different cell types. In certainembodiments, a cytotoxic Shiga cell-targeting molecule, orpharmaceutical composition of the present invention can be used to killspecific cell types in a mixture of different cell types, such aspre-transplantation tissues. In certain embodiments, a Shiga toxineffector polypeptide, cell-targeting molecule, or pharmaceuticalcomposition of the present invention can be used to kill specific celltypes in a mixture of cell types, such as pre-administration tissuematerial for therapeutic purposes. In certain embodiments, acell-targeting molecule or pharmaceutical composition of the presentinvention can be used to selectively kill cells infected by viruses ormicroorganisms, or otherwise selectively kill cells expressing aparticular extracellular target biomolecule, such as a cell surfacebiomolecule. The Shiga toxin effector polypeptides, cell-targetingmolecules, and pharmaceutical compositions of the present invention havevaried applications, including, e.g., uses in depleting unwanted celltypes from tissues either in vitro or in vivo, uses in modulating immuneresponses to treat graft versus host, uses as antiviral agents, uses asanti-parasitic agents, and uses in purging transplantation tissues ofunwanted cell types.

In certain embodiments, certain Shiga toxin effector polypeptides,cell-targeting molecules, and pharmaceutical compositions of the presentinvention, alone or in combination with other compounds orpharmaceutical compositions, can show potent cell-kill activity whenadministered to a population of cells, in vitro or in vivo in a subjectsuch as in a patient in need of treatment. By targeting the delivery ofenzymatically active Shiga toxin A Subunit effector polypeptides and/orT-cell epitopes using high-affinity binding regions to specific celltypes, cell-kill activities can be restricted to specifically andselectively killing certain cell types within an organism, such ascertain cancer cells, neoplastic cells, malignant cells, non-malignanttumor cells, and/or infected cells.

The present invention provides a method of killing a cell in a patientin need thereof, the method comprising the step of administering to thepatient at least one cell-targeting molecule of the present invention ora pharmaceutical composition thereof.

In certain embodiments, the cell-targeting molecule of the presentinvention or pharmaceutical compositions thereof can be used to kill acancer cell in a patient by targeting an extracellular biomolecule foundphysically coupled with a cancer or tumor cell. The terms “cancer cell”or “cancerous cell” refers to various neoplastic cells which grow anddivide in an abnormally accelerated and/or unregulated fashion and willbe clear to the skilled person. The term “tumor cell” includes bothmalignant and non-malignant cells. Generally, cancers and/or tumors canbe defined as diseases, disorders, or conditions that are amenable totreatment and/or prevention. The cancers and tumors (either malignant ornon-malignant) which are comprised of cancer cells and/or tumor cellswhich may benefit from methods and compositions of the invention will beclear to the skilled person. Neoplastic cells are often associated withone or more of the following: unregulated growth, lack ofdifferentiation, local tissue invasion, angiogenesis, and metastasis.The diseases, disorders, and conditions resulting from cancers and/ortumors (either malignant or non-malignant) which may benefit from themethods and compositions of the present invention targeting certaincancer cells and/or tumor cells will be clear to the skilled person.

Certain embodiments of the cell-targeting molecules and compositions ofthe present invention may be used to kill cancer stem cells, tumor stemcells, pre-malignant cancer-initiating cells, and tumor-initiatingcells, which commonly are slow dividing and resistant to cancertherapies like chemotherapy and radiation. For example, acute myeloidleukemias (AMLs) may be treated with the present invention by killingAML stem cells and/or dormant AML progenitor cells (see e.g. Shlush L etal., Blood 120: 603-12 (2012)). Cancer stem cells often overexpress cellsurface targets, such as, e.g., CD44, CD200, and others listed herein,which can be targets of certain binding regions of certain embodimentsof the cell-targeting molecules of the present invention (see e.g.Kawasaki B et al., Biochem Biophys Res Commun 364:778-82 (2007); Reim Fet al., CancerRes 69: 8058-66 (2009)).

Because of the Shiga toxin A Subunit based mechanism of action,compositions of matter of the present invention may be more effectivelyused in methods involving their combination with, or in complementaryfashion with other therapies, such as, e.g., chemotherapies,immunotherapies, radiation, stem cell transplantation, and immunecheckpoint inhibitors, and/or effective againstchemoresistant/radiation-resistant and/or resting tumor cells/tumorinitiating cells/stem cells. Similarly, compositions of matter of thepresent invention may be more effectively used in methods involving incombination with other cell-targeted therapies targeting other than thesame epitope on, non-overlapping, or different targets for the samedisease disorder or condition.

Certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to killan immune cell (whether healthy or malignant) in a patient by targetingan extracellular biomolecule found physically coupled with an immunecell.

It is within the scope of the present invention to utilize acell-targeting molecule of the present invention, or pharmaceuticalcomposition thereof, for the purposes of purging patient cellpopulations (e.g. bone marrow) of malignant, neoplastic, or otherwiseunwanted T-cells and/or B-cells and then reinfusing the T-cell and/orB-cells depleted material into the patient (see e.g. van Heeckeren W etal., Br J Haematol 132: 42-55 (2006); (see e.g. Alpdogan O, van denBrink M, Semin Oncol 39: 629-42 (2012)).

It is within the scope of the present invention to utilize thecell-targeting molecule of the present invention, or pharmaceuticalcomposition thereof, for the purposes of ex vivo depletion of T cellsand/or B-cells from isolated cell populations removed from a patient. Inone non-limiting example, the cell-targeting molecule of the inventioncan be used in a method for prophylaxis of organ and/or tissuetransplant rejection wherein the donor organ or tissue is perfused priorto transplant with a cytotoxic, cell-targeting molecule of the inventionor a pharmaceutical composition thereof in order to purge the organ ofdonor T-cells and/or B-cells (see e.g. Alpdogan O, van den Brink M,Semin Oncol 39: 629-42 (2012)).

It is also within the scope of the present invention to utilize thecell-targeting molecule of the invention, or pharmaceutical compositionthereof, for the purposes of depleting T-cells and/or B-cells from adonor cell population as a prophylaxis against graft-versus-hostdisease, and induction of tolerance, in a patient to undergo a bonemarrow and or stem cell transplant (see e.g. van Heeckeren W et al., BrJ Haematol 132: 42-55 (2006); (see e.g. Alpdogan O, van den Brink M,Semin Oncol 39: 629-42 (2012)).

In certain embodiments of the Shiga toxin effector polypeptide orcell-targeting molecule of the present invention, or pharmaceuticalcompositions thereof, can be used to kill an infected cell in a patientby targeting an extracellular biomolecule found physically coupled withan infected cell.

In certain embodiments of the cell-targeting molecules of the presentinvention, or pharmaceutical compositions thereof, can be used to “seed”a locus within a chordate with non-self, T-cell epitope-peptidepresenting cells in order to activate the immune system to enhancepolicing of the locus. In certain further embodiments of this “seeding”method of the present invention, the locus is a tumor mass or infectedtissue site. In preferred embodiments of this “seeding” method of thepresent invention, the non-self, T-cell epitope-peptide is selected fromthe group consisting of: peptides not already presented by the targetcells of the cell-targeting molecule, peptides not present within anyprotein expressed by the target cell, peptides not present within theproteome or transcriptome of the target cell, peptides not present inthe extracellular microenvironment of the site to be seeded, andpeptides not present in the tumor mass or infect tissue site to betargeting.

This “seeding” method functions to label one or more target cells withina chordate with one or more MHC class I presented T-cell epitopes forrecognition by effector T-cells and activation of downstream immuneresponses. By exploiting the cell internalizing, intracellularlyrouting, and T-cell epitope delivering functions of the cell-targetingmolecules of the present invention, the target cells which display thedelivered T-cell epitope are harnessed to induce recognition of thepresenting target cell by host T-cells and induction of further immuneresponses including target-cell-killing by CTLs. This “seeding” methodof using a cell-targeting molecule of the present invention can providea temporary vaccination-effect by inducing adaptive immune responses toattack the cells within the seeded microenvironment, such as, e.g. atumor mass or infected tissue site, whether presenting a cell-targetingmolecule-delivered T-cell epitope(s) or not. This “seeding” method mayalso induce the breaking of immuno-tolerance to a target cellpopulation, a tumor mass, and/or infected tissue site within a chordate.

Certain methods of the present invention involving the seeding of alocus within a chordate with one or more antigenic and/or immunogenicepitopes may be combined with the administration of immunologicadjuvants, whether administered locally or systemically, to stimulatethe immune response to certain antigens, such as, e.g., theco-administration of a composition of the present invention with one ormore immunologic adjuvants like a cytokine, bacterial product, or plantsaponin. Other examples of immunologic adjuvants which may be suitablefor use in the methods of the present invention include aluminum saltsand oils, such as, e.g., alums, aluminum hydroxide, mineral oils,squalene, paraffin oils, peanut oils, and thimerosal.

Additionally, the present invention provides a method of treating adisease, disorder, or condition in a patient comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of at least one of the cell-targeting molecules of the presentinvention, or a pharmaceutical composition thereof. Contemplateddiseases, disorders, and conditions that can be treated using thismethod include cancers, malignant tumors, non-malignant tumors, growthabnormalities, immune disorders, and microbial infections.Administration of a “therapeutically effective dosage” of a compositionof the present invention can result in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction.

The therapeutically effective amount of a composition of the presentinvention will depend on the route of administration, the type oforganism being treated, and the physical characteristics of the specificpatient under consideration. These factors and their relationship todetermining this amount are well known to skilled practitioners in themedical arts. This amount and the method of administration can betailored to achieve optimal efficacy, and may depend on such factors asweight, diet, concurrent medication and other factors, well known tothose skilled in the medical arts. The dosage sizes and dosing regimenmost appropriate for human use may be guided by the results obtained bythe present invention, and may be confirmed in properly designedclinical trials. An effective dosage and treatment protocol may bedetermined by conventional means, starting with a low dose in laboratoryanimals and then increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

An acceptable route of administration may refer to any administrationpathway known in the art, including but not limited to aerosol, enteral,nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal(e.g. topical administration of a cream, gel or ointment, or by means ofa transdermal patch). “Parenteral administration” is typicallyassociated with injection at or in communication with the intended siteof action, including infraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal administration.

For administration of a pharmaceutical composition of the presentinvention, the dosage range will generally be from about 0.001 to 10milligrams per kilogram (mg/kg), and more, usually 0.001 to 0.5 mg/kg,of the subject's body weight. Exemplary dosages may be 0.01 mg/kg bodyweight, 0.03 mg/kg body weight, 0.07 mg/kg body weight, 0.9 mg/kg bodyweight or 0.1 mg/kg body weight or within the range of 0.01 to 0.1mg/kg. An exemplary treatment regime is a once or twice dailyadministration, or a once or twice weekly administration, once every twoweeks, once every three weeks, once every four weeks, once a month, onceevery two or three months or once every three to 6 months. Dosages maybe selected and readjusted by the skilled health care professional asrequired to maximize therapeutic benefit for a particular patient.

Pharmaceutical compositions of the present invention will typically beadministered to the same patient on multiple occasions. Intervalsbetween single dosages can be, for example, two to five days, weekly,monthly, every two or three months, every six months, or yearly.Intervals between administrations can also be irregular, based onregulating blood levels or other markers in the subject or patient.Dosage regimens for a composition of the present invention includeintravenous administration of 1 mg/kg body weight or 3 mg/kg body weightwith the composition administered every two to four weeks for sixdosages, then every three months at 3 mg/kg body weight or 1 mg/kg bodyweight.

A pharmaceutical composition of the present invention may beadministered via one or more routes of administration, using one or moreof a variety of methods known in the art. As will be appreciated by theskilled worker, the route and/or mode of administration will varydepending upon the desired results. Routes of administration forcell-targeting molecules and pharmaceutical compositions of the presentinvention include, e.g. intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal, or other parenteral routes ofadministration, for example by injection or infusion. For otherembodiments, a cell-targeting molecule or pharmaceutical composition ofthe invention may be administered by a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually, or topically.

Therapeutic cell-targeting molecules or pharmaceutical compositions ofthe present invention may be administered with one or more of a varietyof medical devices known in the art. For example, in one embodiment, apharmaceutical composition of the invention may be administered with aneedleless hypodermic injection device. Examples of well-known implantsand modules useful in the present invention are in the art, includinge.g., implantable micro-infusion pumps for controlled rate delivery;devices for administering through the skin; infusion pumps for deliveryat a precise infusion rate; variable flow implantable infusion devicesfor continuous drug delivery; and osmotic drug delivery systems. Theseand other such implants, delivery systems, and modules are known tothose skilled in the art.

The cell-targeting molecule or pharmaceutical composition of the presentinvention may be administered alone or in combination with one or moreother therapeutic or diagnostic agents. A combination therapy mayinclude a cell-targeting molecule of the present invention, orpharmaceutical composition thereof, combined with at least one othertherapeutic agent selected based on the particular patient, disease orcondition to be treated. Examples of other such agents include, interalia, a cytotoxic, anti-cancer or chemotherapeutic agent, ananti-inflammatory or anti-proliferative agent, an antimicrobial orantiviral agent, growth factors, cytokines, an analgesic, atherapeutically active small molecule or polypeptide, a single chainantibody, a classical antibody or fragment thereof, or a nucleic acidmolecule which modulates one or more signaling pathways, and similarmodulating therapeutic molecules which may complement or otherwise bebeneficial in a therapeutic or prophylactic treatment regimen.

Treatment of a patient with cell-targeting molecule or pharmaceuticalcomposition of the present invention preferably leads to cell death oftargeted cells and/or the inhibition of growth of targeted cells. Assuch, cytotoxic, cell-targeting molecules of the present invention, andpharmaceutical compositions comprising them, will be useful in methodsfor treating a variety of pathological disorders in which killing ordepleting target cells may be beneficial, such as, inter alia, cancer,tumors, other growth abnormalities, immune disorders, and infectedcells. The present invention provides methods for suppressing cellproliferation, and treating cell disorders, including neoplasia,overactive B-cells, and overactive T-cells.

In certain embodiments, the cell-targeting molecules and pharmaceuticalcompositions of the present invention can be used to treat or preventcancers, tumors (malignant and non-malignant), growth abnormalities,immune disorders, and microbial infections. In a further aspect, theabove ex vivo method can be combined with the above in vivo method toprovide methods of treating or preventing rejection in bone marrowtransplant recipients, and for achieving immunological tolerance.

In certain embodiments, the present invention provides methods fortreating malignancies or neoplasms and other blood cell associatedcancers in a mammalian subject, such as a human, the method comprisingthe step of administering to a subject in need thereof a therapeuticallyeffective amount of a cytotoxic cell-targeting molecule orpharmaceutical composition of the present invention.

The cell-targeting molecules and pharmaceutical compositions of thepresent invention have varied applications, including, e.g., uses inremoving unwanted T-cells, uses in modulating immune responses to treatgraft versus host, uses as antiviral agents, uses as antimicrobialagents, and uses in purging transplantation tissues of unwanted celltypes. The cell-targeting molecules and pharmaceutical compositions ofthe present invention are commonly anti-neoplastic agents —meaning theyare capable of treating and/or preventing the development, maturation,or spread of neoplastic or malignant cells by inhibiting the growthand/or causing the death of cancer or tumor cells.

In certain embodiments, the cell-targeting molecule or pharmaceuticalcomposition of the present invention is used to treat a B-cell-, plasmacell- or antibody-mediated disease or disorder, such as for exampleleukemia, lymphoma, myeloma, Human Immunodeficiency Virus-relateddiseases, amyloidosis, hemolytic uremic syndrome, polyarteritis, septicshock, Crohn's Disease, rheumatoid arthritis, ankylosing spondylitis,psoriatic arthritis, ulcerative colitis, psoriasis, asthma, Sjörgren'ssyndrome, graft-versus-host disease, graft rejection, diabetes,vasculitis, scleroderma, and systemic lupus erythematosus.

In another aspect, certain embodiments of the cell-targeting moleculesand pharmaceutical compositions of the present invention areantimicrobial agents —meaning they are capable of treating and/orpreventing the acquisition, development, or consequences ofmicrobiological pathogenic infections, such as caused by viruses,bacteria, fungi, prions, or protozoans.

It is within the scope of the present invention to provide a prophylaxisor treatment for diseases or conditions mediated by T-cells or B-cellsby administering the cell-targeting molecule the present invention, or apharmaceutical composition thereof, to a patient for the purpose ofkilling T-cells or B-cells in the patient. This usage is compatible withpreparing or conditioning a patient for bone marrow transplantation,stem cell transplantation, tissue transplantation, or organtransplantation, regardless of the source of the transplanted material,e.g. human or non-human sources.

It is within the scope of the present invention to provide a bone marrowrecipient for prophylaxis or treatment of host-versus-graft disease viathe targeted cell-killing of host T-cells using a cytotoxiccell-targeting molecule or pharmaceutical composition of the presentinvention.

Certain embodiments of the cell-targeting molecules and pharmaceuticalcompositions of the present invention can be utilized in a method oftreating cancer comprising administering to a patient, in need thereof,a therapeutically effective amount of a cell-targeting molecule and/orpharmaceutical composition of the present invention. In certainembodiments of the methods of the present invention, the cancer beingtreated is selected from the group consisting of: bone cancer (such asmultiple myeloma or Ewing's sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), anduterine cancer.

Certain embodiments of the cell-targeting molecules and pharmaceuticalcompositions of the present invention can be utilized in a method oftreating an immune disorder comprising administering to a patient, inneed thereof, a therapeutically effective amount of the cell-targetingmolecules and/or pharmaceutical composition of the present invention. Incertain embodiments of the methods of the present invention, the immunedisorder is related to an inflammation associated with a diseaseselected from the group consisting of: amyloidosis, ankylosingspondylitis, asthma, Crohn's disease, diabetes, graft rejection,graft-vs.-host disease, Hashimoto's thyroiditis, hemolytic uremicsyndrome, HIV-related diseases, lupus erythematosus, multiple sclerosis,polyarteritis, psoriasis, psoriatic arthritis, rheumatoid arthritis,scleroderma, septic shock, Sjörgren's syndrome, ulcerative colitis, andvasculitis.

Among certain embodiments of the present invention is using the Shigatoxin effector polypeptide or cell-targeting molecule of the presentinvention as a component of a pharmaceutical composition or medicamentfor the treatment or prevention of a cancer, tumor, other growthabnormality, immune disorder, and/or microbial infection. For example,immune disorders presenting on the skin of a patient may be treated withsuch a medicament in efforts to reduce inflammation. In another example,skin tumors may be treated with such a medicament in efforts to reducetumor size or eliminate the tumor completely.

Certain cytotoxic cell-targeting molecules of the present invention, andcompositions thereof, may be used in molecular neurosurgery applicationssuch as immunolesioning and neuronal tracing (see, Wiley R, Lappi D, AdvDrug Deliv Rev 55: 1043-54 (2003), for review). For example, thetargeting domain may be selected or derived from various ligands, suchas neurotransmitters and neuropeptides, which target specific neuronalcell types by binding neuronal surface receptors, such as a neuronalcircuit specific G-protein coupled receptor. Similarly, the targetingdomain may be selected from or derived from antibodies that bindneuronal surface receptors. Because certain Shiga toxin effectorpolypeptides robustly direct their own retrograde axonal transport,certain cell-targeting molecules of the present invention may be used tokill a neuron(s) which expresses the extracellular target at a site ofcytotoxic protein injection distant from the cell body (seeLlewellyn-Smith I et al., J Neurosci Methods 103: 83-90 (2000)). Thesetargeted cytotoxic molecules of the invention that specifically targetneuronal cell types have uses in neuroscience research, such as forelucidating mechanisms of sensations (see e.g. Mishra S, Hoon M, Science340: 968-71 (2013), and creating model systems of neurodegenerativediseases, such as Parkinson's and Alzheimer's (see e.g. Hamlin A et al.,PLoS One e53472 (2013)).

Among certain embodiment of the present invention is a method of using aShiga toxin effector polypeptide, cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention to label or detect the interiors of neoplastic cells and/orimmune cell types. This method may be based on the ability of certaincell-targeting molecules of the present invention to enter specific celltypes and route within cells via retrograde intracellular transport, tothe interior compartments of specific cell types are labeled fordetection. This can be performed on cells in situ within a patient or oncells and tissues removed from an organism, e.g. biopsy material.

Among certain embodiment of the present invention is a method of using aShiga toxin effector polypeptide, cell-targeting molecule,pharmaceutical composition, and/or diagnostic composition of the presentinvention to detect the presence of a cell type for the purpose ofinformation gathering regarding diseases, conditions and/or disorders.The method comprises contacting a cell with a diagnostically sufficientamount of a cell-targeting molecule of the present invention in order todetect the molecule by an assay or diagnostic technique. The phrase“diagnostically sufficient amount” refers to an amount that providesadequate detection and accurate measurement for information gatheringpurposes by the particular assay or diagnostic technique utilized.Generally, the diagnostically sufficient amount for whole organism invivo diagnostic use will be a non-cumulative dose of between 0.001 to 10milligrams of the detection promoting agent linked cell-targetingmolecule of the invention per kg of subject per subject.

Typically, the amount of Shiga toxin effector polypeptide orcell-targeting molecule of the invention used in these informationgathering methods will be as low as possible provided that it is still adiagnostically sufficient amount. For example, for in vivo detection inan organism, the amount of Shiga toxin effector polypeptide,cell-targeting molecule, or pharmaceutical composition of the inventionadministered to a subject will be as low as feasibly possible.

The cell-type specific targeting of cell-targeting molecules of thepresent invention combined with detection promoting agents provides away to detect and image cells physically coupled with an extracellulartarget biomolecule of a binding region of the molecule of the invention.Imaging of cells using the cell-targeting molecules of the presentinvention may be performed in vitro or in vivo by any suitable techniqueknown in the art. Diagnostic information may be collected using variousmethods known in the art, including whole body imaging of an organism orusing ex vivo samples taken from an organism. The term “sample” usedherein refers to any number of things, but not limited to, fluids suchas blood, urine, serum, lymph, saliva, anal secretions, vaginalsecretions, and semen, and tissues obtained by biopsy procedures. Forexample, various detection promoting agents may be utilized fornon-invasive in vivo tumor imaging by techniques such as magneticresonance imaging (MRI), optical methods (such as direct, fluorescent,and bioluminescent imaging), positron emission tomography (PET),single-photon emission computed tomography (SPECT), ultrasound, x-raycomputed tomography, and combinations of the aforementioned (see, Kaur Set al., Cancer Lett 315: 97-111 (2012), for review).

Among certain embodiment of the present invention is a method of using aShiga toxin effector polypeptide, cell-targeting molecule, orpharmaceutical composition of the present invention in a diagnosticcomposition to label or detect the interiors of a hematologic cell,cancer cell, tumor cell, infected cell, and/or immune cell (see e.g.,Koyama Y et al., Clin Cancer Res 13: 2936-45 (2007); Ogawa M et al.,Cancer Res 69: 1268-72 (2009); Yang L et al., Small 5: 235-43 (2009)).Based on the ability of certain cell-targeting molecules of theinvention to enter specific cell types and route within cells viaretrograde intracellular transport, the interior compartments ofspecific cell types are labeled for detection. This can be performed oncells in situ within a patient or on cells and tissues removed from anorganism, e.g. biopsy material.

Diagnostic compositions of the present invention may be used tocharacterize a disease, disorder, or condition as potentially treatableby a related pharmaceutical composition of the present invention. Incertain compositions of matter of the present invention may be used todetermine whether a patient belongs to a group that responds to atherapeutic strategy which makes use of a compound, composition orrelated method of the present invention as described herein or is wellsuited for using a delivery device of the invention.

Diagnostic compositions of the present invention may be used after adisease, e.g. a cancer, is detected in order to better characterize it,such as to monitor distant metastases, heterogeneity, and stage ofcancer progression. The phenotypic assessment of disease disorder orinfection can help prognostic and prediction during therapeutic decisionmaking. In disease reoccurrence, certain methods of the invention may beused to determine if local or systemic problem.

Diagnostic compositions of the present invention may be used to assessresponses to therapies regardless of the type of the type of therapy,e.g. small molecule drug, biological drug, or cell-based therapy. Forexample, certain embodiments of the diagnostics of the invention may beused to measure changes in tumor size, changes in antigen positive cellpopulations including number and distribution, or monitoring a differentmarker than the antigen targeted by a therapy already being administeredto a patient (see Smith-Jones P et al., Nat. Biotechnol 22: 701-6(2004); Evans M et al., Proc. Natl. Acad. Sci. USA 108: 9578-82 (2011)).

In certain embodiments of the method used to detect the presence of acell type may be used to gather information regarding diseases,disorders, and conditions, such as, for example bone cancer (such asmultiple myeloma or Ewing's sarcoma), breast cancer, central/peripheralnervous system cancer (such as brain cancer, neurofibromatosis, orglioblastoma), gastrointestinal cancer (such as stomach cancer orcolorectal cancer), germ cell cancer (such as ovarian cancers andtesticular cancers, glandular cancer (such as pancreatic cancer,parathyroid cancer, pheochromocytoma, salivary gland cancer, or thyroidcancer), head-neck cancer (such as nasopharyngeal cancer, oral cancer,or pharyngeal cancer), hematological cancers (such as leukemia,lymphoma, or myeloma), kidney-urinary tract cancer (such as renal cancerand bladder cancer), liver cancer, lung/pleura cancer (such asmesothelioma, small cell lung carcinoma, or non-small cell lungcarcinoma), prostate cancer, sarcoma (such as angiosarcoma,fibrosarcoma, Kaposi's sarcoma, or synovial sarcoma), skin cancer (suchas basal cell carcinoma, squamous cell carcinoma, or melanoma), uterinecancer, AIDS, amyloidosis, ankylosing spondylitis, asthma, autism,cardiogenesis, Crohn's disease, diabetes, erythematosus, gastritis,graft rejection, graft-versus-host disease, Grave's disease, Hashimoto'sthyroiditis, hemolytic uremic syndrome, HIV-related diseases, lupuserythematosus, lymphoproliferative disorders (including post-transplantlymphoproliferative disorders), multiple sclerosis, myasthenia gravis,neuroinflammation, polyarteritis, psoriasis, psoriatic arthritis,rheumatoid arthritis, scleroderma, septic shock, Sjörgren's syndrome,systemic lupus erythematosus, ulcerative colitis, vasculitis, cellproliferation, inflammation, leukocyte activation, leukocyte adhesion,leukocyte chemotaxis, leukocyte maturation, leukocyte migration,neuronal differentiation, acute lymphoblastic leukemia (ALL), T acutelymphocytic leukemia/lymphoma (ALL), acute myelogenous leukemia, acutemyeloid leukemia (AML), B-cell chronic lymphocytic leukemia (B-CLL),B-cell prolymphocytic lymphoma, Burkitt's lymphoma (BL), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML-BP),chronic myeloid leukemia (CML), diffuse large B-cell lymphoma,follicular lymphoma, hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL),intravascular large B-cell lymphoma, lymphomatoid granulomatosis,lymphoplasmacytic lymphoma, MALT lymphoma, mantle cell lymphoma,multiple myeloma (MM), natural killer cell leukemia, nodal marginalB-cell lymphoma, Non-Hodgkin's lymphoma (NHL), plasma cell leukemia,plasmacytoma, primary effusion lymphoma, pro-lymphocytic leukemia,promyelocytic leukemia, small lymphocytic lymphoma, splenic marginalzone lymphoma, T-cell lymphoma (TCL), heavy chain disease, monoclonalgammopathy, monoclonal immunoglobulin deposition disease,myelodusplastic syndromes (MDS), smoldering multiple myeloma, andWaldenstrom macroglobulinemia.

In certain embodiments, the Shiga toxin effector polypeptides andcell-targeting molecules of the present invention, or pharmaceuticalcompositions thereof, are used for both diagnosis and treatment, or fordiagnosis alone. In some situations, it would be desirable to determineor verify the HLA variant(s) and/or HLA alleles expressed in the subjectand/or diseased tissue from the subject, such as, e.g., a patient inneed of treatment, before selecting a Shiga toxin effector polypeptideor cell-targeting molecule of the invention for use in treatment(s).

Any embodiment of the Shiga toxin effector polypeptide of the presentinvention and cell-targeting molecule of the present invention (e.g.embodiments of embodiment sets #1-11 in the Summary) may be used witheach individual embodiment of the methods of the present invention.

The present invention is further illustrated by the followingnon-limiting examples of 1) Shiga toxin effector polypeptides of thepresent invention, 2) cell-targeting molecules of the present invention,and 3) cytotoxic, cell-targeting molecules of the present inventioncomprising the aforementioned polypeptides and capable of specificallytargeting certain cell types.

EXAMPLES

The following examples demonstrate certain embodiments of the presentinvention. However, it is to be understood that these examples are forillustration purposes only and do not intend, nor should any beconstrued, to be wholly definitive as to conditions and scope of thisinvention. The experiments in the following examples were carried outusing standard techniques, which are well known and routine to those ofskill in the art, except where otherwise described.

The following examples describe several, exemplary, cytotoxic, Shigatoxin A Subunit derived polypeptide scaffolds comprising Shiga toxineffector polypeptides of the present invention. The Shiga toxin effectorpolypeptides in the Examples are de-immunized while retaining catalyticand/or cytotoxic activities.

The following examples also describe several, cytotoxic, cell-targetingmolecules, each molecule comprising a Shiga toxin effector polypeptidelinked, either directly or indirectly, to a cell-targeting bindingregion capable of binding an extracellular part of a target biomoleculephysically associated with a cellular surface of a cell. Exemplary,cytotoxic, cell-targeting molecules described below bound tocell-surface, target biomolecules expressed by targeted, tumorcell-types and entered those targeted cells. The internalized,cell-targeting molecules effectively routed their Shiga toxin effectorpolypeptides to the cytosols of target cells where the Shiga toxineffector polypeptides inactivated ribosomes and subsequently caused theapoptotic death of the targeted cells. The exemplary cell-targetingmolecules of the invention are able to effectively deliver immunogenic,T-cell epitopes to the MHC class I pathway of target cells.

Additionally, some of the exemplary cell-targeting molecules compriseprotease-cleavage resistant, de-immunized, Shiga toxin effectorpolypeptides that exhibited improved in vivo immunogenicity profiles(reductions in antibody responses) as compared to parental cytotoxicmolecules comprising a furin-cleavage resistant, Shiga toxin effectorpolypeptide that had not been further de-immunized by the disruption ofadditional, endogenous epitope regions. Furthermore, these exemplary,protease-cleavage resistant, de-immunized cell-targeting moleculesexhibit improved in vivo tolerability as compared to relatedcell-targeting molecules comprising more protease-cleavage sensitiveShiga toxin effector polypeptide regions.

The Examples below describe certain, Shiga toxin effector polypeptidesof the present invention and their properties. Certain Examples describede-immunized, Shiga toxin effector polypeptides of the present inventionthat comprise embedded, heterologous, CD8+ T-cell epitopes. CertainExamples describe de-immunized, Shiga toxin effector polypeptides of thepresent invention that are furin-cleavage resistant. Certain Examplesdescribe furin-cleavage resistant, de-immunized, Shiga toxin effectorpolypeptides of the present invention that comprise embedded,heterologous, CD8+ T-cell epitopes. Certain Examples describefurin-cleavage resistant, Shiga toxin effector polypeptides of thepresent invention that comprise embedded, heterologous, CD8+ T-cellepitopes with only minimal de-immunization. Furthermore, the Examplesbelow describe certain, cell-targeting molecules of the presentinvention and their properties. Certain Examples describe cell-targetingmolecules of the present invention wherein a Shiga toxin effectorpolypeptide component (1) is de-immunized; (2) is on or proximal to anamino-terminus of a polypeptide component of the cell-targetingmolecule; (3) is furin-cleavage resistant; and/or (4) comprises anembedded or inserted T-cell epitope. Certain Examples describecell-targeting molecules wherein a polypeptide component of thecell-targeting molecule comprises a carboxy-terminal, endoplasmicreticulum retention/retrieval signal motif.

Example 1. Identifying Endogenous, Epitope Regions in Shiga Toxin aSubunit Effector Polypeptides

Polypeptide sequences of the A Subunits from multiple Shiga toxins ofthe Shiga toxin family were analyzed to identify putative, antigenicand/or immunogenic epitopes. This Example shows how antigenic andimmunogenic epitopes can be identified in Shiga toxin A Subunits andrelated polypeptides (see also WO 2015/113005; WO 2015/113007).Computational methods were used to predict antigenic and/or immunogenicepitopes in various Shiga toxin A Subunits, including utilizing publiclyavailable data regarding protein structures. Both B-cell epitopes andCD4+ T-cell epitopes with a potential to elicit immune responses werepredicted in silico. Epitope predictions were validated empirically (seeExample 2, infra; WO 2015/113005; WO 2015/113007).

Linear, B-cell epitopes were predicted for the mature A Subunit ofShiga-like toxin 1 (SLT-1A; SEQ ID NO:1) from the polypeptide sequenceand 3D structural data of Shiga-Like Toxin Chain A (PDB ID: 1DM0_A) byProImmune Inc. (Sarasota, Fla., U.S.) using their REVEAL® system.

In addition, B-cell epitopes were predicted in the polypeptide sequencesof the A Subunits of Shiga toxin (StxA; SEQ ID NO:2), Shiga-like toxin 1(SLT-1A; SEQ ID NO:1), and Shiga-like toxin 2 (Stx2A; SEQ ID NO:3) usingthe BcePred webserver (Saha S, Raghava G, Lecture Notes in Comput Sci3239: 197-204 (2004)), Bepipred Linear Epitope Prediction (Larsen J etal., Immunome Res 2: 2 (2006)), and ElliPro Antibody epitope prediction(Haste Andersen P et al., Protein Sci 15: 2558-67 (2006); Ponomarenko J,Bourne P, BMC Struct Biol 7: 64 (2007)). The various computationalmethods revealed similar predictions for B-cell epitope regions inthree, prototypical, Shiga toxin A Subunits (Tables 1-3).

TABLE 1 B-Cell Epitope Predictions for the Mature, Native A Subunit ofShiga-like Toxin 1 (SEQ ID NO: 1) natively positioned amino acidpositions REVEAL BcePred Bepipred ElliPro 29-35 28-34 27-37 42-48 39-4643-47 58-66 55-61 56-64 57-66  96-103 105-111 100-115  96-110 144-151141-147 147-151 144-153 183-189 181-187 183-185 180-190 211-219 243-251243-257 257-268 261-267 254-268 289-293 285-291 262-293

TABLE 2 B-Cell Epitope Predictions for the Mature, Native A Subunit ofShiga Toxin (SEQ ID NO: 2) natively positioned amino acid positionsREVEAL BcePred Bepipred ElliPro 29-35 28-34 27-37 42-48 39-46 44-4758-66 55-61 56-64 57-66  96-103 105-111 100-115  96-110 144-151 141-147147-151 144-153 183-189 181-187 183-185 180-190 211-219 243-251 243-257257-268 261-267 254-268 289-293 285-291 262-293

TABLE 3 B-Cell Epitope Predictions for the Mature, Native A Subunit ofShiga-like Toxin 2 (SEQ ID NO: 3) natively positioned amino acidpositions BcePred Bepipred ElliPro  3-11  8-14 29-35 28-36 26-37 42-4857-62 56-66 108-115 109-115  96-110 141-156 140-153 179-188 180-191210-218 210-217 240-257 244-258 241-255 262-278 281-297

There were nine, predicted, B-cell epitope regions in SLT-1A which wereidentified by more than one method as overlapping regions (Table 4).

TABLE 4 Putative, B-Cell Epitope Regions in Prototypical, Shiga Toxin ASubunits natively positioned amino acid positions SLT-1A StxA SLT-2A 3-14 27-37 27-37 26-37 39-48 39-48 42-49 55-66 55-66 56-66  96-115 96-115  96-115 141-153 141-153 140-156 180-190 180-190 179-191 210-218243-257 243-257 240-260 254-268 254-268 262-278 285-293 285-293 281-297

In addition, Shiga toxin A Subunits were analyzed using the Epitopiawebserver for predicting B-cell epitopes and immunogenic residues(Rubinstein N et al., BMC Bioinformatics 10: 287 (2009)). Epitopia wasused to identify linear, amino acid residue regions predicted to beimmunogenic in SLT-1A based on an Epitopia score of 4 or 5 (“high”) forthe majority of amino acid residues within a linear, amino acid residueregion.

The Epitopia analysis predicted an immunogenic region occurs from aminoacid residues 1 to 15 in SLT-1A (designated as “Epitope Region 1”, seeTable 5). Based on the Epitopia analysis, the immunogenic epitope region39-48 in SLT-1A (see Table 4) might include position 49 (designated as“Epitope Region 3”, see Table 5). Based on the Epitopia analysis, theimmunogenic epitope region 55-66 in SLT-1A (see Table 4) might includeposition 53 and extend to around position 62-66 (designated as “EpitopeRegion 4”, see Table 5), the epitope region 96-115 in SLT-1A (see Table4) might include position 94 (designated as “Epitope Region 5”, seeTable 5), and the epitope region 180-190 in SLT-1A (see Table 4) mightstart at position 179 and extend to around position 188-190 (designatedas “Epitope Region 7”, see Table 5).

TABLE 5 Prototypical, Shiga Toxin A Subunits Share Ten, Putative, B-CellEpitope Regions Epitope Region natively positioned amino acid positions1  1-15 2 26-37 3 39-49 4 53-66 5  94-115 6 141-153 7 179-190 8 243-2579 254-268 10 285-293

T-cell epitopes were predicted for the mature, A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) by the REVEAL™ Immunogenicity System (IS) T-cellassay performed by ProImmune, Inc. (Sarasota, Fla., U.S.). This assayuses multiple, overlapping peptides from a protein of interest to testfor the elicitation of any immune response by mammalian, CD4+ T-cellsfrom healthy donor cell samples depleted of CD8+ T-cells. ProImmune'sREVEAL™ assay predicted seven, T-cell epitopes in SLT-1A (Table 6).

TABLE 6 Putative, CD4+ T-Cell Epitopes in a Prototypical, Shiga ToxinSubunit natively positioned amino acid positions T-cell epitope #  4-331 34-78 2  77-103 3 128-168 4 160-183 5 236-258 6 274-293 7

All of the ten, predicted, B-cell epitope regions (Table 5) overlappedwith at least one, CD4+ T-cell epitope predicted by the REVEAL™ assay(Table 7).

TABLE 7 B-Cell Epitope Regions in Prototypical, Shiga Toxin A SubunitsOverlap with Predicted, CD4+ T-Cell Epitopes natively positioned aminoacid positions Epitope Region B-Cell Epitope Region T-Cell Epitope(s) 1 1-15  4-33 2 27-37 4-33; 34-78 3 39-48 34-78 4 53-66 34-78 5  94-115 77-103 6 141-153 128-168 7 179-190 160-183 8 243-257 236-258 9 254-268236-258 10 285-293 274-293

In order to improve Shiga toxin-derived polypeptides for therapeutic anddiagnostic applications in chordates, different Shiga toxin A Subuniteffector polypeptides were constructed to be de-immunized andfurin-cleavage resistant, as well as in some instances to comprise anembedded, heterologous, CD8+ T-cell epitope (referred to herein as “CD8+T-cell hyper-immunized”). CD8+ T-cell epitopes were embedded or insertedinto Shiga toxin effector polypeptides by engineering internalsub-regions of Shiga toxin effector polypeptides to comprise aheterologous, T-cell epitope (see e.g. WO 2015/113007). The embedding orinserting of heterologous, T-cell epitopes may be used to disrupt anendogenous, B-cell and/or CD4+ T-cell epitope region(s) in order tofurther de-immunize a Shiga toxin A Subunit derived scaffold (WO2015/113007). All of the predicted, B-cell epitope regions and T-cellepitopes in Table 7 were disrupted and/or deleted individually or incombination in the following examples.

Example 2. Constructing and Testing Exemplary, Shiga Toxin EffectorPolypeptides and Cell-Targeting Molecules of the Present Invention

This example describes the creation and testing of various scaffoldscomprising Shiga toxin effector polypeptides which are de-immunized,such as, e.g., as shown by reductions in antigenicity and/orimmunogenicity relative to other Shiga toxin effector polypeptides. Inaddition, some of the Shiga toxin effector polypeptides of this Exampleare more protease-cleavage resistant than wild-type Shiga toxin effectorpolypeptides and/or comprise embedded or inserted, heterologous, CD8+T-cell epitopes. The Shiga toxin effector polypeptides of this Examplewere tested as components of various, cell-targeting molecules of thepresent invention.

Construction of Exemplary, Shiga Toxin Effector Polypeptides(SLT-1A-Combo(n)) and Cell-Targeting Molecules Comprising the Same(SLT-1A-Combo(n)::scFv-(n))

De-immunized, Shiga toxin A Subunit effector polypeptides were createdand tested in the context of cell-targeting molecules, each comprising acell-targeting, immunoglobulin-type binding region linked to a Shigatoxin effector polypeptide region.

To engineer protease-cleavage resistance into Shiga toxin A Subunitderived polypeptides, the amino acid residue substitutions, R248A and/orR251A, were introduced into Shiga toxin effector polypeptides (see e.g.WO 2015/191764). The R248A and R251A substitutions, either individuallyor in combination, disrupt the furin-cleavage motif at thecarboxy-terminus of the Shiga toxin A1 fragment region and represent oneor more mutations in the minimal, furin-cleavage motif relative to awild-type Shiga toxin A Subunit (see WO 2015/191764).

For this Example, R248A and R251A were introduced into a Shiga toxineffector polypeptide derived from the A subunit of Shiga-like Toxin 1(SLT-1A) comprising amino acids 1-251 of SLT-1A (SEQ ID NO:4). TheSLT-1A 1-251R248A/R251A double mutant (SEQ ID NO:5) is referred toherein as furin-cleavage resistant SLT-1A or more simply “SLT-1A-FR.”The disruption of the minimal, furin-cleavage site R-x-x-R in thefurin-cleavage motif at the carboxy-terminus of the Shiga toxin A1fragment region with R248A and R251A results in decreased cleavage byfurin (see Example 3, infra; WO 2015/191764). The disruption of theminimal furin-cleavage site R-x-x-R in the furin-cleavage motif nativelypositioned from amino acid residues 238 to 257 in StxA and SLT-1A waspredicted to decrease the sensitivity of this region to proteolysis byother proteases such as, e.g., proprotein convertases and highlypromiscuous proteases. In addition, the R248A and/or R251A mutationsdisrupt (1) B-cell epitope region #8, which is natively positioned atamino acid residues 243-259 in StxA and SLT-1A, and (2) a CD4+ T-cellepitope natively positioned at amino acid residues 236-258 in StxA andSLT-1A.

De-immunized, Shiga toxin effector polypeptides were created by addingmultiple, amino acid residue substitutions to disrupt predicted B-celland/or CD4+ T-cell epitope regions, including modifications resulting inembedded, heterologous, CD8+ T-cell epitopes (see Table 8; WO2015/113005; WO 2015/113007). In order to create Shiga toxin effectorpolypeptides that were further de-immunized, SLT-1A-FR (SEQ ID NO:5) wasmodified to include multiple, amino acid residue substitutions todisrupt predicted B-cell and/or CD4+ T-cell epitope regions, includingmodifications resulting in embedded, heterologous, CD8+ T-cell epitopes(see Table 8; WO 2015/113005; WO 2015/113007). Table 8 shows twenty,different, de-immunized, protease-cleavage resistant, Shiga toxin ASubunit effector polypeptides named SLT-1A-combo(n), where n representsan integer such as 0, 1, 2, 3, etc. to denote different variations. Thenumbering of the endogenous epitope regions mentioned in Table 8 is inaccord with the numbering scheme in Tables 6-7. The Shiga toxin effectorpolypeptides in Table 8 were tested as described in later sections.

TABLE 8 Exemplary, De-Immunized, CD8+ T-Cell Hyper-Immunized, ShigaToxin Effector Polypeptides of the Present Invention SLT-1A- combo_((n))name Sequence Summary Description SLT-1A- SEQ ID epitope region 5disrupted by amino acid substitutions combo0 NO: 6 epitope region 5disrupted by embedded, T-cell epitope epitope region 8 disrupted byamino acid substitutions epitope regions 8, 9, and 10 disrupted bytruncation furin-cleavage motif disrupted by R248A/R251A T-cell epitope#6 disrupted by amino acid substitutions T-cell epitopes #6 and #7disrupted by truncation SLT-1A- SEQ ID epitope region 4 disrupted byamino acid substitutions combo1 NO: 7 epitope region 4 disrupted byembedded, T-cell epitope epitope region 8 disrupted by amino acidsubstitutions epitope regions 8, 9, and 10 disrupted by truncationfurin-cleavage motif disrupted by R248A/R251A T-cell epitope #2disrupted by amino acid substitutions T-cell epitope #2 disrupted byembedded, T-cell epitope T-cell epitope #6 disrupted by ammo acidsubstitutions T-cell epitopes #6 and #7 disrupted by truncation SLT-1A-SEQ ID epitope region 3 disrupted by amino acid substitution combo2 NO:8 epitope region 4 disrupted by amino acid substitutions epitope region4 disrupted by embedded, T-cell epitope epitope region 8 disrupted byamino acid substitutions epitope regions 8, 9 and 10 disrupted bytruncation furin-cleavage site disrupted by R248A/R251A T-cell epitope#2 disrupted by amino acid substitutions T-cell epitope #2 disrupted byembedded, T-cell epitope T-cell epitope #6 disrupted by amino acidsubstitutions T-cell epitopes #6 and #7 disrupted by truncation SLT-1A-SEQ ID epitope region 4 disrupted by amino acid substitutions combo3 NO:9 epitope region 4 disrupted by embedded, T-cell epitope epitope region8 disrupted by amino acid substitutions epitope regions 8, 9 and 10disrupted by truncation furin-cleavage site disrupted by R248A/R251AT-cell epitope #2 disrupted by amino acid substitutions T-cell epitope#2 disrupted by embedded, T-cell epitope T-cell epitope #6 disrupted byamino acid substitutions T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 4 disrupted by amino acidsubstitutions combo4 NO: 10 epitope region 4 disrupted by embedded,T-cell epitope epitope region 8 disrupted by amino acid substitutionsepitope regions 8, 9 and 10 disrupted by truncation furin-cleavage sitedisrupted by R248A/R251A T-cell epitope #2 disrupted by amino acidsubstitutions T-cell epitope #2 disrupted by embedded, T-cell epitopeT-cell epitope #6 disrupted by amino acid substitutions T-cell epitopes#6 and #7 disrupted by truncation SLT-1A- SEQ ID epitope region 3disrupted by amino acid substitution combo5 NO: 11 epitope region 4disrupted by amino acid substitutions epitope region 4 disrupted byembedded, T-cell epitope epitope region 5 disrupted by amino acidsubstitution epitope region 7 disrupted by amino acid substitutionepitope regions 8, 9 and 10 disrupted by truncation T-cell epitope #2disrupted by amino acid substitutions T-cell epitope #2 disrupted byembedded, T-cell epitope T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 3 disrupted by amino acidsubstitution combo6 NO: 12 epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 7disrupted by amino acid substitution epitope region 8 disrupted by aminoacid substitutions epitope regions 8, 9 and 10 disrupted by truncationT-cell epitope #2 disrupted by amino acid substitutions T-cell epitope#2 disrupted by embedded, T-cell epitope T-cell epitope #4 disrupted byamino acid substitution T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 3 disrupted by amino acidsubstitution combo7 NO: 13 epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 7disrupted by amino acid substitution epitope region 8 disrupted bysubstitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #6 disrupted by amino acid substitutionsT-cell epitopes #6 and #7 disrupted by truncation SLT-1A- SEQ ID epitoperegion 3 disrupted by amino acid substitution combo21 NO: 27 epitoperegion 4 disrupted by amino acid substitutions epitope region 4disrupted by embedded, T-cell epitope epitope region 5 disrupted byamino acid substitution epitope region 7 disrupted by amino acidsubstitution epitope region 8 disrupted by substitutions furin-cleavagesite disrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#3 disrupted by amino acid substitution T-cell epitope #6 disrupted byamino acid substitutions T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 3 disrupted by amino acidsubstitution combo23 NO: 29 epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 7disrupted by amino acid substitution epitope region 8 disrupted bysubstitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #3 disrupted by amino acid substitutionT-cell epitope #6 disrupted by amino acid substitutions T-cell epitopes#6 and #7 disrupted by truncation SLT-1A- SEQ ID epitope region 3disrupted by amino acid substitution combo24 NO: 30 epitope region 4disrupted by amino acid substitutions epitope region 4 disrupted byembedded, T-cell epitope epitope region 5 disrupted by amino acidsubstitution epitope region 7 disrupted by amino acid substitutionepitope region 8 disrupted by substitutions furin-cleavage sitedisrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#4 disrupted by amino acid substitution T-cell epitope #6 disrupted byamino acid substitutions T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 3 disrupted by amino acidsubstitution combo25 NO: 31 epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 7disrupted by amino acid substitution epitope region 8 disrupted bysubstitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #4 disrupted by amino acid substitutionT-cell epitope #6 disrupted by amino acid substitutions T-cell epitopes#6 and #7 disrupted by truncation SLT-1A- SEQ ID epitope region 3disrupted by amino acid substitution combo26 NO: 32 epitope region 4disrupted by amino acid substitutions epitope region 4 disrupted byembedded, T-cell epitope epitope region 5 disrupted by amino acidsubstitution epitope region 7 disrupted by amino acid substitutionepitope region 8 disrupted by substitutions furin-cleavage sitedisrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#6 disrupted by amino acid substitutions T-cell epitopes #6 and #7disrupted by truncation SLT-1A- SEQ ID epitope region 3 disrupted byamino acid substitution combo8 NO: 14 epitope region 4 disrupted byamino acid substitutions epitope region 4 disrupted by embedded, T-cellepitope epitope region 5 disrupted by amino acid substitution epitoperegion 7 disrupted by amino acid substitution epitope region 8 disruptedby substitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #6 disrupted by amino acid substitutionsT-cell epitopes #6 and #7 disrupted by truncation SLT-1A- SEQ ID epitoperegion 3 disrupted by amino acid substitution combo9 NO: 15 epitoperegion 4 disrupted by amino acid substitutions epitope region 4disrupted by embedded, T-cell epitope epitope region 5 disrupted byamino acid substitution epitope region 7 disrupted by amino acidsubstitution epitope region 8 disrupted by substitutions furin-cleavagesite disrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#6 disrupted by amino acid substitutions T-cell epitopes #6 and #7disrupted by truncation SLT-1A- SEQ ID epitope region 3 disrupted byamino acid substitution combo10 NO: 16 epitope region 4 disrupted byamino acid substitutions epitope region 4 disrupted by embedded, T-cellepitope epitope region 5 disrupted by amino acid substitution epitoperegion 6 disrupted by amino acid substitution epitope region 8 disruptedby substitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #4 disrupted by amino acid substitutionT-cell epitope #6 disrupted by amino acid substitutions T-cell epitopes#6 and #7 disrupted by truncation SLT-1A- SEQ ID epitope region 3disrupted by amino acid substitution combo11 NO: 17 epitope region 4disrupted by amino acid substitutions epitope region 4 disrupted byembedded, T-cell epitope epitope region 5 disrupted by amino acidsubstitution epitope region 7 disrupted by amino acid substitutionepitope region 8 disrupted by substitutions furin-cleavage sitedisrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#6 disrupted by amino acid substitutions T-cell epitopes #6 and #7disrupted by truncation SLT-1A- SEQ ID epitope region 1 disrupted byamino acid substitution combo12 NO: 18 epitope region 3 disrupted byamino acid substitution epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 6disrupted by amino acid substitution epitope region 8 disrupted bysubstitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #4 disrupted by amino acid substitutionT-cell epitope #6 disrupted by amino acid substitutions T-cell epitopes#6 and #7 disrupted by truncation SLT-1A- SEQ ID epitope region 3disrupted by amino acid substitution combo13 NO: 19 epitope region 4disrupted by amino acid substitutions epitope region 4 disrupted byembedded, T-cell epitope epitope region 7 disrupted by amino acidsubstitutions epitope region 7 disrupted by embedded T-cell epitopeepitope region 8 disrupted by substitutions furin-cleavage sitedisrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#4 disrupted by amino acid substitutions T-cell epitope #6 disrupted byamino acid substitutions T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 3 disrupted by amino acidsubstitution combo14 NO: 20 epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 6disrupted by amino acid substitution epitope region 7 disrupted by aminoacid substitution epitope region 8 disrupted by substitutionsfurin-cleavage site disrupted by R248A/R251A epitope regions 8, 9 and 10disrupted by truncation T-cell epitope #2 disrupted by amino acidsubstitutions T-cell epitope #2 disrupted by embedded, T-cell epitopeT-cell epitope #4 disrupted by amino acid substitution T-cell epitope #6disrupted by amino acid substitutions T-cell epitopes #6 and #7disrupted by truncation SLT-1A- SEQ ID epitope region 1 disrupted byamino acid substitution combo15 NO: 21 epitope region 3 disrupted byamino acid substitution epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 6disrupted by amino acid substitution epitope region 8 disrupted bysubstitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #2 disruptedby amino acid substitutions T-cell epitope #2 disrupted by embedded,T-cell epitope T-cell epitope #4 disrupted by amino acid substitutionT-cell epitope #6 disrupted by amino acid substitutions T-cell epitopes#6 and #7 disrupted by truncation SLT-1A- SEQ ID epitope region 1disrupted by amino acid substitution combo16 NO: 22 epitope region 3disrupted by amino acid substitution epitope region 4 disrupted by aminoacid substitutions epitope region 4 disrupted by embedded, T-cellepitope epitope region 5 disrupted by amino acid substitution epitoperegion 6 disrupted by amino acid substitution epitope region 7 disruptedby amino acid substitution epitope region 8 disrupted by substitutionsfurin-cleavage site disrupted by R248A/R251A epitope regions 8, 9 and 10disrupted by truncation T-cell epitope #2 disrupted by amino acidsubstitutions T-cell epitope #2 disrupted by embedded, T-cell epitopeT-cell epitope #4 disrupted by amino acid substitution T-cell epitope #6disrupted by amino acid substitutions T-cell epitopes #6 and #7disrupted by truncation SLT-1A- SEQ ID epitope region 1 disrupted byamino acid substitutions combo17 NO: 23 epitope region 3 disrupted byamino acid substitution epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 6disrupted by amino acid substitution epitope region 7 disrupted by aminoacid substitution epitope region 8 disrupted by substitutionsfurin-cleavage site disrupted by R248A/R251A epitope regions 8, 9 and 10disrupted by truncation T-cell epitope #1 disrupted by amino acidsubstitution T-cell epitope #2 disrupted by amino acid substitutionsT-cell epitope #2 disrupted by embedded, T-cell epitope T-cell epitope#4 disrupted by amino acid substitution T-cell epitope #6 disrupted byamino acid substitutions T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 1 disrupted by amino acidsubstitutions combo18 NO: 24 epitope region 3 disrupted by amino acidsubstitution epitope region 4 disrupted by amino acid substitutionsepitope region 4 disrupted by embedded, T-cell epitope epitope region 5disrupted by amino acid substitution epitope region 6 disrupted by aminoacid substitution epitope region 7 disrupted by amino acid substitutionepitope region 8 disrupted by substitutions furin-cleavage sitedisrupted by R248A/R251A epitope regions 8, 9 and 10 disrupted bytruncation T-cell epitope #1 disrupted by amino acid substitution T-cellepitope #2 disrupted by amino acid substitutions T-cell epitope #2disrupted by embedded, T-cell epitope T-cell epitope #4 disrupted byamino acid substitution T-cell epitope #6 disrupted by amino acidsubstitutions T-cell epitopes #6 and #7 disrupted by truncation SLT-1A-SEQ ID epitope region 1 disrupted by amino acid substitution combo19 NO:25 epitope region 3 disrupted by amino acid substitution epitope region4 disrupted by amino acid substitutions epitope region 4 disrupted byembedded, T-cell epitope epitope region 5 disrupted by amino acidsubstitution epitope region 6 disrupted by amino acid substitutionepitope region 7 disrupted by substitutions epitope region 8 disruptedby substitutions furin-cleavage site disrupted by R248A/R251A epitoperegions 8, 9 and 10 disrupted by truncation T-cell epitope #1 disruptedby amino acid substitution T-cell epitope #2 disrupted by amino acidsubstitutions T-cell epitope #2 disrupted by embedded, T-cell epitopeT-cell epitope #4 disrupted by amino acid substitution T-cell epitope #5disrupted by amino acid substitution T-cell epitope #6 disrupted byamino acid substitutions T-cell epitopes #6 and #7 disrupted bytruncation SLT-1A- SEQ ID epitope region 3 disrupted by amino acidsubstitution combo20 NO: 26 epitope region 4 disrupted by amino acidsubstitutions epitope region 4 disrupted by embedded, T-cell epitopeepitope region 5 disrupted by amino acid substitution epitope region 6disrupted by amino acid substitution epitope region 8 disrupted bysubstitutions epitope region 8 disrupted by inserted, T-cell epitopefurin-cleavage site disrupted by R248A/R251A epitope regions 8, 9 and 10disrupted by truncation T-cell epitope #2 disrupted by amino acidsubstitutions T-cell epitope #2 disrupted by embedded, T-cell epitopeT-cell epitope #4 disrupted by amino acid substitution T-cell epitope #6disrupted by amino acid substitutions T-cell epitope #6 disrupted byinserted, T-cell epitope T-cell epitopes #6 and #7 disrupted bytruncation

Each of the de-immunized, Shiga toxin effector polypeptidesSLT-1A-combo(n) (SEQ ID NOs: 6-21, 23-27, and 29-32) comprise acombination of one or more de-immunized sub-regions with one or moresub-regions that comprises an embedded, heterologous, CD8+ T-cellepitope. Most of these polypeptides also have a disrupted, minimal,furin-cleavage motif at the carboxy-terminal of their Shiga toxin A1fragment sub-regions (SEQ ID NOs: 6-10, 13-21, 23-27, and 29-32).Computational analysis in silico predicted (1) that at least two, B-cellepitopes present in the wild-type Shiga toxin StxA or SLT-1A wereeliminated for all of the Shiga toxin effector polypeptides combo(n)referred to in Table 8, and (2) that no new, B-cell epitopes werecreated in any of the Shiga toxin effector polypeptides combo(n) listedin Table 8. The properties and functional consequences of many of themodifications to individual sub-regions in Shiga toxin effectorpolypeptides combo(n) are described in Example 3, WO 2015/113005, WO2015/113007, and WO 2015/191764.

Polynucleotides encoding Shiga toxin A Subunit effector polypeptidecombination constructs were created and fused to a cell-targeting,immunoglobulin-type binding region encoding constructs using techniquesknown to the skilled worker. The resulting polynucleotides encodedcell-targeting molecules, each a polypeptide comprising (1) acombination, Shiga toxin effector polypeptide region SLT-1A-combo(n),(2) a cell-targeting binding region “scFv-(n),” where n represents aninteger such as 1, 2, 3, etc. to denote different scFvs, and (3) alinker known in the art positioned between the Shiga toxin effectorpolypeptide region and the binding region.

Using a bacterial expression system known in the art, thesepolynucleotides were used to produce at least twenty-seven, combination,de-immunized, Shiga toxin effector polypeptides (Table 8; SEQ ID NOs:6-21, 23-27, and 29-32), in the context of one or more cell-targetingmolecules. After being linked to a cell-targeting binding region,twenty-six of the twenty-seven scaffolds SLT-1A-combo(n) produced astable, full-length, catalytically active, cell-targeting molecule.However, the cell-targeting molecule SLT-1A-combo18::scFv-1 (SEQ IDNO:54) showed evidence of instability because SLT-1A-combo18::scFv-1degradation was observed by sodium dodecyl sulfate (SDS), polyacrylamidegel electrophoretic (SDS-PAGE) analysis of gels loaded and ran withpreparations of SLT-1A-combo18::scFv-1 and a molecular weight ladder asa reference.

A. Testing the Ribosome Inhibition Activities of Exemplary,Cell-Targeting Molecules Comprising Shiga Toxin Effector PolypeptidesSLT-1A-Combo(n)

The enzymatic activities of various, combination, de-immunized,protease-cleavage resistant, Shiga toxin effector polypeptides weretested in the context of a cell-targeting molecule using an in vitro,ribosome inhibition assay known to the skilled worker (TNT® QuickCoupled Transcription/Translation Kit, Promega Corp., Madison, Wis.,U.S.). This cell-free, in vitro, protein translation assay was used todetermine the ribosome inactivation capabilities ofSLT-1A-combo10:scFv-1 (SEQ ID NO:47), SLT-1A-combo16::scFv-1 (SEQ IDNO:52), SLT-1A-combo19::scFv-1 (SEQ ID NO:55), SLT-1A-combo0::scFv-2(SEQ ID NO:57), SLT-1A-combo2::scFv-2 (SEQ ID NO:58),SLT-1A-combo3::scFv-2 (SEQ ID NO:59), SLT-1A-combo4::scFv-2 (SEQ IDNO:60), and SLT-1A-combo13::scFv-2 (SEQ ID NO:62).

The ribosome activity reaction was prepared according to manufacturer'sinstructions. A series of 10-fold dilutions of the cell-targetingmolecule to be tested was prepared in an appropriate buffer and a seriesof identical TNT® reaction mixture components were created for eachdilution. Each sample in the dilution series was combined with each ofthe TNT® reaction mixtures along with Luciferase T7 Control DNA (PromegaCorp., Madison, Wis., U.S.). The test samples were incubated for 1.5hours at 30 degrees Celsius (° C.). After the incubation, LuciferaseAssay Reagent (Promega Corp., Madison, Wis., U.S.) was added to all testsamples and the amount of luciferase protein translation was measured byluminescence according to manufacturer's instructions. Three positivecontrols were used: the wild-type, SLT-1A1 fragment (SLT-1A1-WT) (SEQ IDNO:4) and two cell-targeting molecules SLT-1A-FR::scFv-1 (SEQ ID NO:34)and SLT-1A-FR::scFv-2 (SEQ ID NO:35) comprising the protease-cleavageresistant, Shiga toxin effector polypeptide SLT-1A-FR (SEQ ID NO:5) (seeWO 2015/191764).

The level of protein synthesis inhibition was determined by non-linearregression analysis of log-transformed concentrations of totalcell-targeting molecule versus relative luminescence units. Usingstatistical software (GraphPad Prism, San Diego, Calif., U.S.), the halfmaximal inhibitory concentration (IC₅₀) value was calculated for eachsample using the Prism software function of log(inhibitor) vs. response(three parameters) [Y=Bottom+((Top−Bottom)/(1+10{circumflex over( )}(X−Log IC₅₀)))] under the heading dose-response-inhibition. The IC₅₀for each sample was calculated and is shown in Table 9. In this assay,measurements of the inhibition of protein synthesis represent theribosome inactivation activity of the sample molecule, which is onemetric of the catalytic activity of a Shiga toxin effector polypeptideor a Shiga toxin A Subunit. As reported in the Examples, a moleculeexhibiting an IC₅₀ within 10-fold of an IC₅₀ exhibited by a referencemolecule is considered to exhibit ribosome inhibition activitycomparable to that reference molecule. As reported in the Examples, amolecule exhibiting an IC₅₀ less than or within 10 percent of an IC₅₀exhibited by a reference molecule is considered to exhibit ribosomeinhibition activity equivalent to that reference molecule.

TABLE 9 Combination, De-Immunized, Protease-Cleavage Resistant, ShigaToxin Effector Polypeptides Exhibited Ribosome Inhibition ActivityComparable to a Wild-Type Shiga toxin A1 Fragment Ribosome InhibitionProtein Sample IC₅₀ (pM) SLT-1A-combo10::scFv-1 2.8SLT-1A-combo16::scFv-1 3.3 SLT-1A-combo19::scFv-1 2.2 SLT-1A-FR::scFv-14.0 SLT-1A-combo0::scFv-2 4.8 SLT-1A-combo2::scFv-2 3.1SLT-1A-combo3::scFv-2 7.6 SLT-1A-combo4::scFv-2 6.5SLT-1A-combo13::scFv-2 3.2 SLT-1A-FR::scFv-2 5.9 SLT-1A1-WT 4.8

The ribosome inactivation activities of all the Shiga toxin effectorpolypeptides combo(n) tested were comparable to the catalytic activityof a wild-type Shiga toxin A1 fragment (SEQ ID NO:4) and/or a SLT-1A-FRpolypeptide (SEQ ID NO:5) as a component of a cell-targeting molecule(Table 9; FIG. 2). The ribosome inactivation activities ofSLT-1A-combo10::scFv-1, SLT-1A-combo16::scFv-1, SLT-1A-combo19::scFv-1,SLT-1A-combo0::scFv-2, SLT-1A-combo2::scFv-2, SLT-1A-combo4::scFv-1, andSLT-1A-combo13::scFv2 were equivalent to the catalytic activity of awild-type Shiga toxin A1 fragment (SEQ ID NO:4) and/or a SLT-1A-FRpolypeptide (SEQ ID NO:5) as a component of a cell-targeting molecule(Table 9; FIG. 2).

These results demonstrate that the ribosome inhibition activities ofcertain, exemplary, combination, de-immunized, protease-cleavageresistant, Shiga toxin effector polypeptides in the context of acell-targeting molecule were comparable to the catalytic activity of awild-type Shiga toxin A1 fragment (SEQ ID NO:4) alone or the Shiga toxineffector polypeptide SLT-1A-FR (SEQ ID NO:5) in the context of acell-targeting molecule (Table 9; FIG. 2). Thus, the catalyticactivities of certain, exemplary, combination, de-immunized,protease-cleavage resistant, Shiga toxin effector polypeptides in thecontext of a cell-targeting molecule appeared comparable in this assayto the catalytic activity of a wild-type Shiga toxin A Subunit (Table 9;FIG. 2).

B. Testing the Targeted Cytotoxicity of Exemplary, Cell-TargetingMolecules Comprising Shiga Toxin Effector Polypeptides SLT-1A-Combo(n)

The potency and specificity of cytotoxicity were tested for combination,de-immunized, Shiga toxin effector polypeptides SLT-1A-combo(n) of thepresent invention as scaffolds for building various cell-targetingmolecules. The cytotoxic activities of exemplary, cell-targetingmolecules comprising a combination, de-immunized, protease-cleavageresistant, Shiga toxin effector polypeptide SLT-1A-combo(n) weredetermined using a target biomolecule positive cell-kill assay known tothe skilled worker. This target positive cell-kill assay was used todetermine the cytotoxic activities of various cell-targeting molecules,each comprising a cell-targeting binding region scFv-(n) geneticallyfused to one of the combination, Shiga toxin effector polypeptidesSLTA-1A-combo(n) (SEQ ID NOs: 6-32) (see Table 8 for a representativesubset of such molecules) to form the cell-targeting molecules SEQ IDNOs: 43-81.

The cytotoxicities of cell-targeting molecules comprising de-immunized,protease-cleavage resistant, Shiga toxin effector polypeptides weredetermined using cells expressing, at a cellular surface, significantamounts of the appropriate, extracellular target biomolecule, such as, atarget of the binding region scFv-1-8. The immunoglobulin-derivedbinding regions scFv-1, scFv-2, scFv-3, scFv-4, scFv-5, scFv-6, scFv-7,scFv-8, and scFv-9 each binds with high affinity to a human targetbiomolecule physically coupled to the cellular surfaces of certain humancells. The cells used in this Example were immortalized, human tumorcells available from the ATCC (Manassas Va., U.S.), National CancerInstitute of the U.S. (Frederick, Md., U.S.), and/or DSZM (Braunschweig,Del.). The cells referred to below were H929, Daudi,NCI-ADR/RES(expressing HER-2 from a transfected vector), HCC1419,MDA-MB-231, MOLP-8, ST486, HDLM-2, and L1236 or more simply cell-linesA, B, C, D, E, F, G, H, and I, respectively. Using a method known to theskilled worker, cells from cell-line C used in this Example weretransfected with an expression vector and made to express a significantamount of cell-surface HER-2.

The cell-kill assay was performed as follows. Certain, human tumor,cell-line cells were plated (at about 2 to 8×10³ cells per well) in 20microliter (μL) cell culture medium in 384-well plates. A series of10-fold dilutions of the cell-targeting molecules to be tested wereprepared in an appropriate buffer, and 5 μL of the dilutions or buffercontrol were added to the plated cells. Control wells containing onlycell culture medium were used for baseline correction. The cell sampleswere incubated with the cell-targeting molecules or just buffer forthree or five days at 37° C. and in an atmosphere of five percent carbondioxide (CO₂). The total cell survival or percent viability wasdetermined using a luminescent readout using the CellTiter-Glo®Luminescent Cell Viability Assay (G7573, Promega Corp., Madison, Wis.,U.S.) according to the manufacturer's instructions.

The Percent Viability of experimental wells was calculated using thefollowing equation: (Test RLU−Average Media RLU)/(Average Cells onlyRLU−Average Media RLU)*100. The logarithm of the cell-targeting moleculeconcentration versus Percent Viability was plotted in Prism (GraphPadPrism, San Diego, Calif., U.S.), and log (inhibitor) versus response (3parameter) analysis was used to determine the half-maximal cytotoxicconcentration (CD₅₀) value for the tested cell-targeting molecule. TheCD₅₀ for each sample was calculated and is shown in Table 10. When CD₅₀values could not be calculated based on the shape of the curve over theconcentrations tested, then a maximum CD₅₀ value was noted as beingbeyond the maximum tested value, e.g., greater than 100 nM (“>100.000nM”) or 200 nM (“>200.000 nM”), for samples which did not kill 50% ofthe cells at the highest, tested, sample concentration, e.g., 100.000 nMor 200.000 nM. If the cell viability in the assay was approximately 50%at the highest tested sample concentration, then the CD₅₀ value for thatmolecule was noted in Table 10 as being approximately the maximumconcentration tested where the cell viability was approximately 50%,e.g., “˜100.000 nM.” As reported in the Examples, a molecule exhibitinga CD₅₀ within 10-fold of a CD₅₀ exhibited by a reference molecule isconsidered to exhibit cytotoxic activity comparable to that referencemolecule.

TABLE 10 Exemplary, De-Immunized, Protease-Cleavage Resistant, ShigaToxin Effector Polypeptides Exhibited Potent Cytotoxicity CD₅₀ (nM) CD₅₀(nM) Cell-Targeting Molecule scFv-1 target scFv-1 target Set 1 - scFv-1positive cell line A positive cell line B Experiment 1SLT-1A-combo10::scFv-1 0.025 0.079 SLT-1A-combo16::scFv-1 0.032 0.115SLT-1A-combo19::scFv-1 0.032 0.157 SLT-1A-FR::scFv-1 0.014 0.045Experiment 2 SLT-1A-combo17::scFv-1 0.040 ~100.000SLT-1A-combo18::scFv-1 >100.000 >100.000 SLT-1A-FR::scFv-1 0.007 0.048SLT-1A-WT >100.000 >100.000 Experiment 3 SLT-1A-combo7::scFv-1 0.026SLT-1A-FR::scFv-1 0.017 Cell-Targeting Molecule CD₅₀ (nM) CD₅₀ (nM) CD₅₀(nM) Set 1, Experiment 3 scFv-1 target scFv-1 target scFv-1 target(cont'd) positive cell line F positive cell line G negative cell line HSLT-1A-combo7::scFv-1 0.386 0.133 >100.000 SLT-1A-FR::scFv-1 0.2830.099 >100.000 scFv-1 target scFv-1 target Experiment 4 positive cellline A positive cell line G SLT-1A-combo1::scFv-1 0.017 0.024SLT-1A-combo10::scFv-1 0.027 0.068 SLT-1A-combo12::scFv-1 0.067 0.182SLT-1A-combo15::scFv-1 0.054 0.094 SLT-1A-combo17::scFv-1 0.088 0.162SLT-1A-FR::scFv-1 0.013 0.020 scFv-1 target scFv-1 target scFv-1 targetExperiment 5 positive cell line A positive cell line F positive cellline G SLT-1A-combo7::scFv-1 0.01 0.12 0.02 SLT-1A-combo8::scFv-1 0.010.14 0.04 SLT-1A-combo9::scFv-1 0.01 0.33 0.13 SLT-1A-combo11::scFv-10.02 0.67 0.55 scFv-1 target scFv-1 target scFv-1 target Experiment 6positive cell line A positive cell line B positive cell line FSLT-1A-combo10::scFv-1 0.03 0.25 0.84 SLT-1A-combo20::scFv-1 0.17 0.793.21 scFv-2 target scFv-2 target scFv-2 target Set 2 - scFv-2 positivecell line A positive cell line B positive cell line G Experiment 7SLT-1A-combo0::scFv-2 1.52 17.2 SLT-1A-combo3::scFv-2 0.68 3.18SLT-1A-combo4::scFv-2 0.08 0.44 SLT-1A-FR::scFv-2 0.01 0.06 Experiment 8SLT-1A-combo2::scFv-2 2.206 1.70 0.798SLT-1A-combo13::scFv-2 >100.000 >100.000 >100.000 SLT-1A-FR::scFv-20.119 0.171 0.112 Experiment 9 SLT-1A-combo22::scFv-2 0.059 0.259 0.110SLT-1A-FR::scFv-2 0.041 0.172 0.070 scFv-3 scFv-3 target scFv-3 targetscFv-3 target Experiment 10 positive cell line B positive cell line Gnegative cell line A scFv-3::SLT-1A-combo5 73.80 5.48 >200.00scFv-3::SLT-1A-combo6 54.70 1.01 >200.00 scFv-3::SLT-1A 119.000.79 >200.00 scFv-4 scFv-4 target scFv-4 target scFv-4 target Experiment11 positive cell line C positive cell line D negative cell line ESLT-1A-combo7::scFv-4 0.045 0.059 >200.000 SLT-1A-combo14::scFv-4 0.0650.079 >200.000 SLT-1A-FR::scFv-4 0.012 0.018 >200.000 Set 3 - scFv-5scFv-5 target scFv-5 target Experiment 12 positive cell line I positivecell line H SLT-1A-combo7::scFv-5 0.037 0.029 SLT-1A-FR::scFv5 0.0120.012 Experiment 13 SLT-1A-combo8::scFv-5 0.064 SLT-1A-combo9::scFv-50.376 SLT-1A-combo11::scFv-5 ~100.000 SLT-1A-FR::scFv-5 0.011 scFv-6target scFv-6 target Set 4 - scFv-6 positive cell line I positive cellline H Experiment 14 SLT-1A-combo7::scFv-6 0.037 0.157 SLT-1A-FR::scFv-60.075 0.038 Experiment 15 SLT-1A-combo8::scFv-6 1.699SLT-1A-combo9::scFv-6 ~100.000 SLT-1A-FR::scFv-6 0.204 Experiment 16SLT-1A-combo7::scFv-6 0.240 SLT-1A-combo21::scFv-6 1.900SLT-1A-combo23::scFv-6 0.260 SLT-1A-combo24::scFv-6 0.310SLT-1A-FR::scFv-6 0.130 scFv-6 target Experiment 17 positive cell line ISLT-1A-combo7::scFv-6 0.500 SLT-1A-combo25::scFv-6 0.100SLT-1A-combo26::scFv-6 0.730 scFv-8 scFv-8 scFv-8 Experiment 18 positivecell line B negative cell line A SLT-1A-combo0::scFv-8 0.077 >100.000SLT-1A-FR::scFv-8 0.440 >100.000

The CD₅₀ values for exemplary, SLT-1A-combo scaffold-based,cell-targeting molecules are shown in Table 10 and associated cell-killassay data is shown in FIGS. 3-10. These results show the cell-killassay results for at least thirty-four, unique, cell-targeting moleculeseach comprising a combination, de-immunized, Shiga toxin effectorpolypeptide SLT-1A-combo(n) selected from at least twenty-six Shigatoxin effector polypeptides (SEQ ID NOs: 6-27 and 29-32). Thirty-two ofthirty-four combination, de-immunized, Shiga toxin effector polypeptidesSLT-1A-combo(0-12, 14-17, 19-21, 23-27, and 29-32) effectuatedcytotoxicities, in the context of a cell-targeting molecule,characterized by CD₅₀ values equal to or less than 100 nM, whereas twocell-targeting molecules, comprising either SLT-1A-combo13 (SEQ IDNO:19) and SLT-1A-combo18 (SEQ ID NO:24), exhibited cytotoxicitiescharacterized by CD₅₀ values greater than 100 nM toward the twocell-lines tested (Table 10; FIGS. 6-7).

The results reported in Table 10 show that the cell-targeting moleculescomprising SLT-1A-combo0, SLT-1A-combo1, SLT-1A-combo4, SLT-1A-combo7,SLT-1A-combo8, SLT-1A-combo9, SLT-1A-combo10, SLT-1A-combo1l,SLT-1A-combo12, SLT-1A-combo14, SLT-1A-combo15, SLT-1A-combo16,SLT-1A-combo17, SLT-1A-combo19, or SLT-1A-combo25 displayed potentcytotoxicities characterized by CD₅₀ values of 0.2 nM or less dependingon the cell-line tested (Table 10; FIGS. 3-10). The cytotoxicities ofmost of the Shiga toxin effector polypeptides combo(n) tested as acomponent of a cell-targeting molecule were comparable to thecytotoxicities of a SLT-1A-FR polypeptide (SEQ ID NO:5) as a componentof a related, cell-targeting molecule (Table 10; FIGS. 3-7; 9-10). Forexample, the cytotoxicities to at least one cell-type tested for atleast one cell-targeting molecule comprising one of the Shiga toxineffector polypeptides combo(0-2, 4, 7-8, 10, 12, 14-19, or 23-26) werecomparable to the cytotoxicities of related, cell-targeting moleculescomprising a SLT-1A-FR polypeptide (SEQ ID NO:5) (Table 10; FIGS. 3-7;9-10).

The specificity of the cytotoxicities of exemplary, cell-targetingmolecules comprising certain, combination, de-immunized, Shiga toxineffector polypeptides SLT-1A-combo(n) were determined usingtarget-negative cell-kill assays known to the skilled worker. The targetbiomolecule negative cell-kill assay is identical to the target-positivecell-kill assay except for the cell type used. The target negativecell-kill assay was performed using cells which do not expresssignificant amounts of the extracellular target biomolecule bound withhigh-affinity by the respective binding region scFv-(n) of thecell-targeting molecule being tested. The cytotoxic specificities ofexemplary, cell-targeting molecules comprising certain, combination,de-immunized, Shiga toxin effector polypeptide scaffolds SLT-1A-combo(n)were determined by comparing the results from target positive cell-killassays with results from target-negative cell-kill assays (see e.g.Table 10; FIGS. 8-9). Cytotoxic, cell-targeting molecules comprisingde-immunized, Shiga toxin effector polypeptides SLT1-A-combo(5-7, 14, or21) or SLT-1A-FR (SEQ ID NO:5) did not kill comparable percentages oftarget negative cells as compared to target positive cells at the same,cell-targeting molecule concentrations (Table 10; FIGS. 8-9).

C. Testing for Caspase Activation Induced by Exemplary, Cell-TargetingMolecules Comprising Shiga Toxin Effector Polypeptides SLT-1A-Combo(n)

Apoptosis is a programmed cell death involving the degradation ofcellular components by caspases. Apoptosis can be detected by monitoringthe activity of the effector capsases 3 and 7, such as, e.g., theactivation state of either caspase 3 or caspase 7. The caspaseactivation of exemplary, cell-targeting molecules comprisingcombination, de-immunized, protease-cleavage resistant, Shiga toxineffector polypeptides SLT-1A-combo(n) of the present invention weredetermined using a target biomolecule positive cell, caspase activityassay known to the skilled worker. The method of the target positivecell, caspase activity assay is analogous to the target-positivecell-kill assay described above.

Shiga toxin catalytic activity can induce apoptosis of Shigatoxin-intoxicated, mammalian cells via an intrinsic pathway (see e.g.,Inward C et al., J Infect 30: 213-8 (1995); Fujii J et al., Infect Immun71: 2724-35 (2003); Jandhyala D et al., Curr Top Microbiol Immunol 357:41-65 (2012); Tesh V, Curr Top Microbiol Immunol 357: 137-78 (2012)).Caspase activation can be used as a readout for the activation ofapoptotic mechanisms leading to cell death (see Pop C, Salvesen G, JBiol Chem 284: 21777-81 (2009); Nicholls S, Hyman B, Methods Enzymol544: 251-69 (2014)). The potency and specificity of caspase activationinduced by the combination, de-immunized, protease-cleavage resistant,Shiga toxin effector polypeptides SLT-1A-combo7 and SLT-1A-combo14 inthe context of a cell-targeting molecules were determined as follows.

The caspase activation of cell-targeting molecules comprisingde-immunized, protease-cleavage resistant, Shiga toxin effectorpolypeptide scaffolds were determined using cells expressing, at acellular surface, significant amounts of an extracellular targetbiomolecule of the binding region scFv-1 or scFv-6.

The caspase activation assay was performed as follows. Certain, humantumor, cell-line cells were plated (at about 2-8×10³ cells per well) in20 microliter (μL) cell culture medium in 384-well plates. A series of10-fold dilutions of the cell-targeting molecules to be tested wereprepared in an appropriate buffer, and 5 L of the dilutions or buffercontrol were added to the plated cells. Control wells containing onlycell culture medium were used for baseline correction. The cell sampleswere incubated with the cell-targeting molecules or just buffer for18-20 hours at 37° C. and in an atmosphere of five percent CO₂. Thecaspase activation was determined using a luminescent readout using theCaspase-Glo 3/7® Luminescent Cell Viability Assay (Promega Corp.,Madison, Wis., U.S.) according to the manufacturer's instructions.

The amount of caspase activation in experimental wells was calculatedusing the following equation: ((Test RLU−Average Media RLU)/(AverageCells RLU−Average Media RLU))*100. The logarithm of the cell-targetingmolecule concentration versus Caspase Activation was plotted in Prism(GraphPad Prism, San Diego, Calif., U.S.) and log (agonist) versusresponse (3 parameter) analysis was used for each, tested,cell-targeting molecule to calculate the half-maximal effectiveconcentration (EC₅₀) value for caspase activation in the assay (FIGS.11-12; Table 11). The maximum caspase activity percentage (the percentactivation over the “cells only” control measurements) for eachexperiment (maximum activity) was calculated using caspase activationmeasurements from samples with “cells only” as the baseline (Table 11).The EC₅₀ and maximum activity for caspase activation for exemplarycell-targeting molecules are shown in Table 11.

TABLE 11 Exemplary, De-Immunized, Protease-Cleavage Resistant,Cell-Targeting Molecules Induced Caspase Activation caspase activitymaximum caspase activity maximum EC₅₀ (nM) activity (%) EC₅₀ (nM)activity (%) scFv-1 target scFv-1 target Cell-Targeting Moleculepositive cell line A positive cell line G SLT-1A-combo7::scFv-1 0.072562 0.066 245 SLT-1A-combo14::scFv-1 0.083 534 0.094 245SLT-1A-FR::scFv-1 0.054 561 0.045 250 scFv-7 target scFv-7 targetpositive cell line H positive cell line I SLT-1A-combo7::scFv-7 2.410219 0.210 216 SLT-1A-FR::scFv-7 0.860 335 0.050 234

The caspase activity in target cells induced by the Shiga toxin effectorpolypeptide combos 7 and 14, each tested as a component of acell-targeting molecule, were comparable to the caspase activity inducedby the SLT-1A-FR polypeptide (SEQ ID NO:5) as a component of a related,cell-targeting molecule (SEQ ID NO:34 and SEQ ID NO:39, respectively)for most of the cell-lines tested (Table 11; FIGS. 11-12).

D. Endogenous Epitope Disrupting Mutations in Shiga Toxin EffectorPolypeptides

This Example shows that Shiga toxin A Subunit effector polypeptides canbe de-immunized with certain truncations and combinations of amino acidresidue substitutions. Deletions and/or amino acid substitutions weremade in the putative, B-cell and/or T-cell epitopes of Shiga toxineffector polypeptides derived from the A Subunit of Shiga-like Toxin 1(SLT-1A) as listed in Table 12. In this Examples and in WO 2015/113005,many mutations have been empirically tested for effect(s) on the Shigatoxin effector function of various Shiga toxin effector polypeptides andcell-targeting molecules. Table 12 summarizes the results described inthe Examples and in WO 2015/113005 where an amino acid substitution orcombination of amino acid substitutions did not prevent the exhibitionof a potent level of Shiga toxin effector function. Table 12 uses theepitope region numbering scheme described in the Example 1 (see Table 7,supra), and lists any change in B-cell epitopes predicted by BcePredsoftware.

TABLE 12 Substitutions and Combinations of Substitutions EmpiricallyVerified Not to Prevent Exhibition of a Potent Shiga Toxin EffectorFunction(s) natively positioned amino acid positions Epitope EpitopeRibosome Cytotox- Region Substitution(s) Prediction Inhibition icity 1K1M/K11A no change YES YES 1 S8I no change YES YES 1 T9I no change YESYES 2 S33I eliminated YES YES 3 T45I eliminated YES YES 3 S45Ieliminated YES YES 4 D53A no change YES YES 4 R55A no change YES YES 4D58A eliminated YES YES 4 D58F eliminated YES YES 4 P59A eliminated YESYES 4 E60I eliminated YES YES 4 E60R no change YES YES 4 E61A eliminatedYES YES 4 G62A eliminated YES YES 5 D94A/S96I no change YES YES 7 D183Ano change YES YES 7 D184A eliminated YES YES 7 D184F eliminated YES YES7 R188A eliminated YES YES 7 D183A/D184A/R188A eliminated YES YESimmuno- R205A eliminated YES YES genic residue 4 and 5 E60I/G110Aeliminated YES YES 4 and 6 E60I/G147A eliminated YES YES 3 and 7T45I/R188A eliminated YES YES 3 and 7 S45I/R188A eliminated YES YES 2,4, and 6 S33I/G110A/G147A eliminated YES YES 3, 4, and 6T45I/G110A/G147A eliminated YES YES 3, 4, and 6 S45I/G110A/G147Aeliminated YES YES 4. 5, and 6 D58A/G110A/G147A eliminated YES YES 4, 5,and 6 E60I/G110A/G147A eliminated YES YES 3, 4, 5, and 6 T45I/D58A/E60I/eliminated YES YES G110A/G147A 3, 4, 5, and 6 S45I/D58A/E60I/ eliminatedYES YES G110A/G147A 3, 4, 5, and 6 T45I/D58A/E60I/ eliminated YES YESG62A/G110A/G147A 3, 4, 5, and 6 S45I/D58A/E60I/ eliminated YES YESG62A/G110A/G147A 3, 4, 5, 6, T45I/G110A/ eliminated YES YES and 7G147A/D183A/ D184A/R188A 3, 4, 5, 6, S45I/G110A/ eliminated YES YES and7 G147A/D183A/ D184A/R188A 4, 5, 6, and 7 D58A/G110A/ eliminated YES YESG147A/S186A 4, 5, 6, and 7 D58A/G110A/ eliminated YES YES G147A/G187A 4,5, 6, and 7 D58A/G110A/ eliminated YES YES G147A/R188A 4, 5, 6, and 7D58A/G110A/ eliminated YES YES G147A/S189A 4, 5, 6, and 7 D58A/G110A/eliminated YES YES G147A/S186A/ R188A 4, 5, 6, and 7 D58A/G110A/eliminated YES YES G147A/G187A/ R188A 2, 3, 4, 5, S33I/S45I/D58A/eliminated YES YES and 6 G110A/G147A 3, 4, 5, 6, T45I/D58A/E60I/eliminated YES YES and 7 G110A/G147A/ D183A/D184A/ R188A 3, 4, 5, 6,S45I/D58A/E60I/ eliminated YES YES and 7 G110A/G147A/ D183A/D184A/ R188A3, 4, 5, 6, T45I/D58A/E60I/ eliminated YES YES and 7 G62A/G110A/G147A/D183A/ D184A/R188A 3, 4, 5, 6, S45I/D58A/E60I/ eliminated YES YESand 7 G62A/G110A/ G147A/D183A/ D184A/R188A 4, 5, 6, 7, D58A/G110A/eliminated YES YES and 8 G147A/R188A/ C242S/R248A/ R251A 5 and 8T104N/A105L/ eliminated YES YES T107P/L108M/ S109V/G110A/ D111T/R248A/R251A 4 and 8 V54I/R55L/I57F/ eliminated YES P59F/E60T/E61L/ R248A/R251A3 and 8 S43N/G44L/ eliminated YES YES T45V/G46P/ D47M/N48V/ L49A/F5017A51V/R248A/ R251A 3 and 8 S43N/G44L/ eliminated YES YES S45V/G46P/D47M/N48V/ L49A/F50T/ A51V/R248A/ R251A 3, 4, and 8 T45I/V54I/R55L/eliminated YES YES I57F/P59F/E60T/ E61L/C242S/ R248A/R251A 3, 4, and 8S45I/V54I/R55L/ eliminated YES YES I57F/P59F/E60T/ E61L/C242S/R248A/R251A 4 and 8 D53N/V54L/ eliminated YES YES R55V/G56P/ I57M/D58V/P59A/E60T/ E61V/R248A/ R251A 3, 4, 5, and 7 T45I/V54I/R55L/ eliminatedYES I57F/P59F/E60T/ E61L/G110A/ R188A 3, 4, 5, and 7 S45I/V54I/R55L/eliminated YES I57F/P59F/E60T/ E61L/G110A/ R188A 4, 5, 6, and 8D58A/G110A/ eliminated YES YES G147A/R188A/C242S 3, 4, 5, 6,T45I/V54I/R55L/ eliminated YES and 7 I57F/P59F/E60T/ E61L/G110A/G147A/R188A 3, 4, 5, 6, S45I/V54I/R55L/ eliminated YES and 7I57F/P59F/E60T/ E61L/G110A/ G147A/R188A 3, 4, 5, 7, T45I/V54I/R55L/eliminated YES and 8 I57F/P59F/E60T/ E61L/G110A/ R188A/C242S/R248A/R251A 3, 4, 5, 7, S45I/V54I/R55L/ eliminated YES and 8I57F/P59F/E60T/ E61L/G110A/ R188A/C242S/ R248A/R251A 3, 4, 5, 7,T45I/V54I/R55L/ eliminated YES and 8 I57F/P59F/E60T/ E61L/R84A/G110A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7, S45I/V54I/R55L/ eliminatedYES and 8 I57F/P59F/E60T/ E61L/R84A/ G110A/R188A/ C242S/R248A/ R251A 3,4, 5, 7, T45I/V54I/R55L/ eliminated YES and 8 I57F/P59F/E60T/ E61L/V88A/G110A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7, S45I/V54I/R55L/ eliminatedYES and 8 I57F/P59F/E60T/ E61L/V88A/ G110A/R188A/ C242S/R248A/ R251A 3,4, 5, 7, T45I/V54I/R55L/ eliminated YES and 8 I57F/P59F/E60T/E61L/G110A/ D141A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7, S45I/V54I/R55L/eliminated YES and 8 I57F/P59F/E60T/ E61L/G110A/ D141A/R188A/C242S/R248A/ R251A 3, 4, 5, 7, T45I/V54I/R55L/ eliminated YES and 8I57F/P59F/E60T/ E61L/G110A/ V154A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7,S45I/V54I/R55L/ eliminated YES and 8 I57F/P59F/E60T/ E61L/G110A/V154A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7, T45I/V54I/R55L/ eliminatedYES and 8 I57F/P59F/E60T/ E61L/G110A/ R188A/D198A/ C242S/R248A/ R251A 3,4, 5, 7, S45I/V54I/R55L/ eliminated YES and 8 I57F/P59F/E60T/E61L/G110A/ R188A/D198A/ C242S/R248A/ R251A 3, 4, 5, 7, T45I/V54I/R55L/eliminated YES and 8 I57F/D58V/P59F/ E60T/E61L/ G110A/R188A/C242S/R248A/ R251A 3, 4, 5, 7, S45I/V54I/R55L/ eliminated YES and 8I57F/D58V/P59F/ E60T/E61L/ G110A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7,T45I/D53G/V54I/ eliminated YES and 8 R55L/I57F/P59F/ E60T/E61L/G110A/R188A/ C242S/R248A/ R251A 3, 4, 5, 7, S45I/D53G/V54I/ eliminatedYES and 8 R55L/I57F/P59F/ E60T/E61L/ G110A/R188A/ C242S/R248A/ R251A 3,4, 5, 6, T45I/V54I/R55L/ eliminated YES YES and 8 I57F/P59F/E60T/E61L/G110A/ G147A/C242S/ R248A/R251A 3, 4, 5, 6, S45I/V54I/R55L/eliminated YES YES and 8 I57F/P59F/E60T/ E61L/G110A/ G147A/C242S/R248A/R251A 3, 4, 5, 7, T45I/D53G/V54I/ eliminated YES and 8R55L/I57F/D58V/ P59F/E60T/E61L/ G110A/R188A/ C242S/R248A/ R251A 3, 4, 5,7, S45I/D53G/V54I/ eliminated YES and 8 R55L/I57F/D58V/ P59F/E60T/E61L/G110A/R188A/ C242S/R248A/ R251A 3, 4, 7, and 8 K1A/S45I/V54I/ eliminatedYES R55L/I57F/P59F/ E60T/E61L/ G110A/G147A/ C242S/R248A/ R251A 3, 4, 7,and 8 S45I/V54I/R55L/ eliminated YES I57F/P59F/E60T/ E61L/T180G/T181I/D183G/ D184F/L185D/ S186F/G187T/ C242S/R248A/ R251A 3, 4, 5, 6,T45I/V54I/R55L/ eliminated YES 7, and 8 I57F/P59F/E60T/ E61L/G110A/G147A/R188A/ C242S/R248A/ R251A 3, 4, 5, 6, S45I/V54I/R55L/ eliminatedYES 7, and 8 I57F/P59F/E60T/ E61L/G110A/ G147A/R188A/ C242S/R248A/ R251A1, 3, 4, 5, K1A/T45I/V54I/ eliminated YES 6, 7, and 8 R55L/I57F/P59F/E60T/E61L/ G110A/G147A/ R188A/C242S/ R248A/R251A 1, 3, 4, 5,K1A/S45I/V54I/ eliminated YES 6, 7, and 8 R55L/I57F/P59F/ E60T/E61L/G110A/G147A/ R188A/C242S/ R248A/R251A 1, 3, 4, 5, T4I/T45I/V54I/eliminated YES YES 6, 7, and 8 R55L/I57F/P59F/ E60T/E61L/ G110A/G147A/R188A/C242S/ R248A/R251A 1, 3, 4, 5, T4I/S45I/V54I/ eliminated YES YES6, 7, and 8 R55L/I57F/P59F/ E60T/E61L/ G110A/G147A/ R188A/C242S/R248A/R251A 1, 3, 4, 5, K1A/T4I/T45I/ eliminated YES 6, 7, and 8V54I/R55L/I57F/ P59F/E60T/E61L/ G110A/G147A/ R188A/C242S/ R248A/R251A 1,3, 4, 5, K1A/T4I/S45I/ eliminated YES 6, 7, and 8 V54I/R55L/I57F/P59F/E60T/E61L/ G110A/G147A/ R188A/C242S/ R248A/R251A 1, 3, 4, 5,T4I/T45I/D58A/ eliminated YES YES 6, 7, and 8 E60I/G62A/ G110A/G147A/D183A/D184A/ R188A/C242S/ R248A/R251A 1, 3, 4, 5, T4I/S45I/D58A/eliminated YES YES 6, 7, and 8 E60I/G62A/ G110A/G147A/ D183A/D184A/R188A/C242S/ R248A/R251A 3, 4, 5, 6, T45I/V54I/R55L/ eliminated YES and8 I57F/P59F/E60T/ E61L/G110A/ G147A/▾ins 11 residues/C242S/ R248A/R251A3, 4, 5, 6, S45I/V54I/R55L/ eliminated YES and 8 I57F/P59F/E60T/E61L/G110A/ G147A/▾ins 11 residues/C242S/ R248A/R251A

In epitope regions 1-5 and 7-8, different amino acid substitutions havebeen made and tested (see Table 12). In epitope region #1 (see Table 7),the lysine natively located at position 1 in the mature A Subunits ofShiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO: 2) wasmutated to alanine (KIA) and methionine (KIM)). In epitope region #1,the threonine natively located at position 4 in the mature A Subunits ofShiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ ID NO:2) wasmutated to isoleucine (T4I). In epitope region #1, the aspartatenatively located at position 6 in the mature A Subunits of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toarginine (D6R). In epitope region #1, the serine natively located atposition 8 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (S8I). Inepitope region #1, the threonine natively located at position 9 in themature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to isoleucine (T9I) and to valine (T9V). Inepitope region #1, the lysine natively located at position 11 in themature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to alanine (K11A) and to histidine (K11H). Inepitope region #1, the threonine natively located at position 12 in themature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to lysine (T12K).

In epitope region #2 (see Table 7), the serine natively located atposition 33 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (S33I).

In epitope region #3 (see Table 7), the serine natively located atposition 43 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)was mutated to asparagine (S43N). In epitope region #3, the glycinenatively located at position 44 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) was mutated to leucine (G44L). In epitope region#3, the serine natively located at position 45 in the mature A Subunitof Shiga-like toxin 1 (SEQ ID NO:1) was mutated to valine (S45V) and toisoleucine (S45I). In epitope region #3, the threonine natively locatedat position 45 in the mature A Subunit of Shiga-like toxin 1 (SEQ IDNO:2) was mutated to valine (T45V) and to isoleucine (T45I). In epitoperegion #3, the glycine natively located at position 46 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) was mutated to proline(G46P). In epitope region #3, the aspartate natively located at position47 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) wasmutated to glycine (D47G) and to methionine (D47M). In epitope region#3, the asparagine natively located at position 48 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) was mutated to valine (N48V)and to phenylalanine (N48F).

In epitope region #4 (see Table 7), the aspartate natively located atposition 53 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D53A), glycine(D53G), and asparagine (D53N). The D53 residue was predicted by theEpitopia webserver to be solvent exposed and have an immunogenicityscale value of 5 or “high.” In epitope region #4, the valine nativelylocated at position 54 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine(V54I). In epitope region #4, the arginine natively located at position55 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shigatoxin (SEQ ID NO:2) was mutated to alanine (R55A), to valine (R55V), andto leucine (R55L). In epitope region #4, the glycine natively located atposition 56 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to proline (G56P). In epitoperegion #4, the isoleucine natively located at position 57 in the matureA Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to methionine (D57M) and to phenylalanine (D57F). Inepitope region #4, the aspartate natively located at position 58 in themature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to alanine (D58A), to valine (D58V), and tophenylalanine (D58F). In epitope region #4, the proline natively locatedat position 59 in the mature A Subunit of Shiga-like toxin 1 (SEQ IDNO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (P59A). Inepitope region #4, the glutamate natively located at position 60 in themature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to isoleucine (E601), to threonine (E60T), andto arginine (E60R). In epitope region #4, the glutamate natively locatedat position 61 in the mature A Subunit of Shiga-like toxin 1 (SEQ IDNO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine (E61A), tovaline (E61V), and to leucine (E61L). In epitope region #4, the glycinenatively located at position 62 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toalanine (G62A).

In epitope region #5 (see Table 7), the aspartate natively located atposition 94 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D94A). In epitoperegion #5, the serine natively located at position 96 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to isoleucine (S961). In epitope region #5, thethreonine natively located at position 104 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to asparagine (T104N). In epitope region #5, the alaninenatively located at position 105 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toleucine (A105L). In epitope region #5, the threonine natively located atposition 107 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to proline (T107P). In epitoperegion #5, the leucine natively located at position 108 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to methionine (L108M). In epitope region #5, theserine natively located at position 109 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to valine (S109V). In epitope region #5, the glycine nativelylocated at position 110 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine(G110A). In epitope region #5, the aspartate natively located atposition 111 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to threonine (D111T). Inepitope region #5, the serine natively located at position 112 in themature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to valine (S112V).

In epitope region #6 (see Table 7), the aspartate natively located atposition 141 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D141A). In epitoperegion #6, the glycine natively located at position 147 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to alanine (G147A).

In epitope region #7 (see Table 7), the arginine natively located atposition 179 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R179A). This R179residue was predicted by the Epitopia webserver to be exposed and havean immunogenicity value of 5 or “high.” In epitope region #7 (see Table7), the threonine natively located at position 180 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to glycine (T180G). In epitope region #7 (see Table7), the threonine natively located at position 181 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to isoleucine (T1811). In epitope region #7 (see Table7), the aspartate natively located at position 183 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO: 1) and Shiga toxin (SEQ IDNO:2) was mutated to alanine (D183A) and to glycine (D183G). In epitoperegion #7, the aspartate natively located at position 184 in the matureA Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to alanine (D184A) or phenylalanine (D184F). Inepitope region #7 (see Table 7), the leucine natively located atposition 185 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to valine (L185V) or aspartate(L185D). In epitope region #7, the serine natively located at position186 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) andShiga toxin (SEQ ID NO:2) was mutated to alanine (S186A) and tophenylalanine (S186F). In epitope region #7, the glycine nativelylocated at position 187 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine(G187A) and to threonine (G187T). In epitope region #7, the argininenatively located at position 188 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toalanine (R188A) and to leucine (R188L). In epitope region #7, the serinenatively located at position 189 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toalanine (S189A).

In epitope region #8 (see Table 7), the arginine natively located atposition 248 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R248A). In epitoperegion #8, the arginine natively located at position 251 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to alanine (R251A).

The leucine natively located at position 49 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to alanine (L49A). This L49 residue was predicted by theEpitopia webserver to be solvent exposed and have an immunogenicityvalue of 4 and is present in T-cell epitope #2 (see Table 7). Theglutamate natively located at position 198 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to alanine (D198A). This D198 residue was predicted by theEpitopia webserver to be solvent exposed and have an immunogenicityvalue of 5 or “high.” The arginine natively located at position 205 inthe mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to alanine (R205A). This R205 residue waspredicted by the Epitopia webserver to be solvent exposed and have animmunogenicity value of 5 or “high.”

In T-cell epitopes #1 through #6, different amino acid substitutionshave been made and tested (see Table 12).

In T-cell epitope #1 (see Table 7), the threonine natively located atposition 4 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine (T4I), theaspartate natively located at position 6 in the mature A Subunits ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to arginine (D6R), the serine natively located at position 8 inthe mature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1) and Shigatoxin (SEQ ID NO:2) was mutated to isoleucine (S8I), the threoninenatively located at position 9 in the mature A Subunits of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toisoleucine (T9I) and to valine (T9V), the lysine natively located atposition 11 in the mature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (K11A) and tohistidine (K11H), the threonine natively located at position 12 in themature A Subunits of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to lysine (T12K), and the serine nativelylocated at position 33 in the mature A Subunits of Shiga-like toxin 1(SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to isoleucine(S33I).

In T-cell epitope #2 (see Table 7), the serine natively located atposition 43 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)was mutated to asparagine (S43N); the glycine natively located atposition 44 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)was mutated to leucine (G44L); the serine natively located at position45 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) wasmutated to valine (S45V) and to isoleucine (S451); the glycine nativelylocated at position 46 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) was mutated to proline (G46P); the aspartate nativelylocated at position 47 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) was mutated to glycine (D47G) and to methionine (D47M);the asparagine natively located at position 48 in the mature A Subunitof Shiga-like toxin 1 (SEQ ID NO:1) was mutated to valine (N48V) and tophenylalanine (N48F); the phenylalanine natively located at position 50in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shigatoxin (SEQ ID NO:2) was mutated to alanine (F50T); the alanine nativelylocated at position 51 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to valine(A51V); the aspartate natively located at position 53 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to alanine (D53A), glycine (D53G), and asparagine(D53N); the valine natively located at position 56 in the mature ASubunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ IDNO:2) was mutated to leucine (V54L); the arginine natively located atposition 55 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R55A), to valine(R55V), and to leucine (R55L); the glycine natively located at position56 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shigatoxin (SEQ ID NO:2) was mutated to proline (G56P); the isoleucinenatively located at position 57 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated tomethionine (D57M) and to phenylalanine (D57F); the aspartate nativelylocated at position 58 in the mature A Subunit of Shiga-like toxin 1(SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated to alanine(D58A), to valine (D58V), and to phenylalanine (D58F); the prolinenatively located at position 59 in the mature A Subunit of Shiga-liketoxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) was mutated toalanine (P59A); the glutamate natively located at position 60 in themature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to isoleucine (E60I), to threonine (E60T), andto arginine (E60R); the glutamate natively located at position 61 in themature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to alanine (E61A), to valine (E61V), and toleucine (E61L); the glycine natively located at position 62 in themature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin(SEQ ID NO:2) was mutated to alanine (G62A).

In T-cell epitope #3 (see Table 7), the aspartate natively located atposition 94 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (D94A), and theserine natively located at position 96 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to isoleucine (S96I).

In T-cell epitope #4 (see Table 7), the glycine natively located atposition 147 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (G147A).

In T-cell epitope #5 (see Table 7), the arginine natively located atposition 179 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R179A), thethreonine natively located at position 180 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to glycine (T180G), the threonine natively located at position181 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) andShiga toxin (SEQ ID NO:2) was mutated to isoleucine (T1811), and theaspartate natively located at position 183 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to alanine (D183A) and to glycine (D183G).

In T-cell epitope #6 (see Table 7), the cysteine natively located atposition 242 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to serine (C242S), thearginine natively located at position 248 in the mature A Subunit ofShiga-like toxin 1 (SEQ ID NO:1) and Shiga toxin (SEQ ID NO:2) wasmutated to alanine (R248A), and the arginine natively located atposition 251 in the mature A Subunit of Shiga-like toxin 1 (SEQ ID NO:1)and Shiga toxin (SEQ ID NO:2) was mutated to alanine (R251A).

Furthermore, truncating the carboxy-terminus of SLT-1A to amino acids1-251 of SEQ ID NO: 1 removed the last two epitope regions (Table 7, #9and #10), the last CD4+ T-cell epitope (Table 6, #7), and the highestscoring discontinuous, B-cell epitope predicted by ElliPro (289-293). Inaddition, the truncation at position 251 disrupts T-cell epitope #6 andepitope region #8 (Table 7).

An exemplary, de-immunized, Shiga toxin effector polypeptide of thisExample is SLT-1A-combo22 (SEQ ID NO:28), which has seven, amino acidresidue substitutions relative to the wild-type Shiga-like toxin 1 ASubunit (SEQ ID NO:1) and all of these substitutions were predicted todisrupt endogenous epitopes. In accord with the labels in Table 8, theShiga toxin effector polypeptide SLT-1A-combo22 comprises a substitutiondisrupting epitope region 4; a substitution disrupting epitope region 5;a substitution disrupting epitope region 6; a substitution disruptingepitope region 7; substitutions disrupting epitope region 8; epitoperegions 8, 9, and 10 disrupted by truncation; and substitutionsdisrupting the furin-cleavage site at the carboxy-terminus of the A1fragment-derived region. In addition, SLT-1A-combo22 comprises asubstitution disrupting T-cell epitope #2, a substitution disruptingT-cell epitope #4, substitutions disrupting epitope #6, and T-cellepitopes #6 and #7 disrupted by truncation.

E. Testing for Reductions in Antigenicities of Exemplary, Shiga ToxinEffector Polypeptides SLT-1A-Combo(n) Using ELISA and Western BlotAssays

Routine methods may be used to evaluate the relative antigenicities ofShiga toxin effector polypeptides in the context of cell-targetingmolecules (see e.g. WO 2015/113005; WO 2015/113007). The antigenicitiesof exemplary, cell-targeting molecules comprising certain, combination,de-immunized, protease-cleavage resistant, Shiga toxin effectorpolypeptides SLT-1A-combo(n) was assessed by Western blots and ELISAsusing both polyclonal and monoclonal antibodies that bind withhigh-affinity to the wild-type Shiga-like toxin A1 fragment (SLT-1A1).The cell-targeted molecule SLT-1A-FR::scFv-1 (SEQ ID NO:34) was used asa reference molecule.

The Western analyses used herein determined relative antigenicity underdenaturing conditions whereas the ELISA analyses used herein measuredrelative antigenicity under native protein folding conditions.

For Western analyses, exemplary, cell-targeting molecules comprising theShiga toxin effector polypeptides SLT-1A-combo7, SLT-1A-combo10, orSLT-1A-combo14 were tested and compared to the results for the referencemolecule SLT-1A-FR::scFv-1. Samples of the aforementioned molecules wereloaded in equal amounts to replicate, 4-20% SDS polyacrylamide gels(Lonza, Basel, CH) and electrophoresed under denaturing conditions. Theresulting gels were either analyzed by Coomassie staining or transferredto polyvinyl difluoride (PVDF) membranes using the iBlot® (LifeTechnologies, Carlsbad, Calif., U.S.) system according to manufacturer'sinstructions. The resulting membranes were probed under standardconditions using the following antibodies: mouse monoclonal α-Stx (mAb1)(BEI NR-867 BEI Resources, Manassas, Va., U.S.; cross reactive with theShiga-like toxin 1 A Subunit), rabbit polyclonal antibody α-SLT-1A(pAb1) (Harlan Laboratories, Inc. Indianapolis, Ind., U.S., customantibody production raised against the wild-type SLT-1A1, and rabbitpolyclonal antibody α-SLT-1A (pAb2) (Genscript, Piscataway, N.J., U.S.,custom antibody production) which was raised against peptides from thewild-type Shiga toxin A1 fragment: RGIDPEEGRFNN (SEQ ID NO: 593) andHGQDSVRVGR (SEQ ID NO: 594). The peptide sequence RGIDPEEGRFNN (SEQ IDNO: 593) is located at amino acids 55-66 and the peptide sequenceHGQDSVRVGR (SEQ ID NO: 594) is located at 214-223 in SLT-1A and StxA.Membrane bound antibodies were detected using standard conditions and,when appropriate, using horseradish peroxidase (HRP)-conjugatedsecondary antibodies (goat anti-rabbit-HRP or goat anti-mouse-HRP,Thermo Scientific, Rockford, Ill., U.S.). FIG. 13 shows Western blotswith the lanes of the gels and/or membranes numbered and the figurelegend indicating by the same respective numbering which Shiga toxineffector polypeptide regions were present in the cell-targeting moleculeloaded into each lane. Coomassie stained lanes are shown as sampleloading controls.

FIG. 13 shows that, under denaturing conditions, exemplarycell-targeting molecules SLT-1A-combo7::scFv-1, SLT-1A-combo10::scFv-1,and SLT-1A-combo14::scFv-1 exhibit decreased antigenicities as comparedto SLT-1A-FR::scFv-1 in this assay. These results demonstrate thatSLT-1A-combo7, SLT-1A-combo10, and SLT-1A-combo14 have decreasedantigenicities as compared to SLT-1A-FR, or by inference to SLT-1A-WT,when linked by the same fashion to the same targeting domain (scFv-1)using the same linker. Additionally, the results shown in FIG. 13suggest that SLT-1A-combo7 has reduced relative antigenicity as comparedto SLT-1A-combo10 and SLT-1A-combo14 in this assay under the conditionstested.

A standard ELISA was used to measure the ability of α-SLT-1A mAb1 torecognize various de-immunized, Shiga toxin effector polypeptides, eachwith multiple epitope regions disrupted, in the context of acell-targeting molecule. The ability of each cell-targeting moleculetested to bind the target biomolecule of its scFv binding region wasutilized in the assay. The wells of Nunc MaxiSorp® plates in phosphatebuffered saline (1×PBS) (Hyclone Brand, Fisher Scientific, Waltham,Mass., U.S.) were coated with recombinant, human target biomolecule ofthe binding region (scFv-1). The plates were incubated overnight at 4°C. The wells were washed with 1×PBS 0.05% Tween-20 (PBS-T), andnon-specific binding was blocked by incubating the wells with 3% milk inPBS-T for one hour at room temperature. Exemplary cell-targetingmolecules were added to the wells, where certain wells received only onecell-targeting molecule comprising only one, combination, de-immunized,Shiga toxin effector polypeptide SLT-1A-combo(n): SLT-1A-combo7::scFv-1,SLT-1A-combo10::scFv-1, or SLT-1A-combo14::scFv-1.

In addition, the cell-targeted molecule SLT-1A-FR::scFv-1 was added tocertain wells as a reference molecule. All cell-targeting molecules wereadded to the wells at a concentration determined to be above the maximumbinding (Bmax) determined previously with an ELISA using Protein Lconjugated to HRP to detect SLT-1A-FR::scFv-1, thus allowing for 100% ofthe available target biomolecules to be bound by the cell-targetingmolecule sample, which is in excess. The plates were incubated at roomtemperature for one hour to allow for cell-targeting molecule to bind totarget biomolecule under non-denaturing conditions. The wells werewashed with PBS-T and then incubated with anti-SxtA mouse monoclonalantibody conjugated to HRP (anti-SLT-1A mAb1-RP) or rabbit polyclonalantibody α-SLT-1A conjugated to HRP (anti-SLT-1A pAb2-RP) or proteinL-HRP (which binds to scFv-1 and was used as a loading control) for 1hour at room temperature. The wells were washed in PBS-T then incubatedwith Pierce TMB Ultra (Thermo Scientific Inc., Rockford, Ill., U.S.).The reactions were stopped with 250 mM hydrochloric acid (HCl). HRPactivity was detected in the wells by adding a chromogenic HRP substrateand then detecting light emission, resulting from chemiluminscence,using a plate reading device measuring absorbance (Abs) of light set tothe wavelength of 450 nanometers (nm).

The measured absorbance values were corrected for background bysubtracting the absorbance values for coated, blocked wells incubatedwith only PBS instead of any cell-targeting molecule sample. Tonormalize the signals from the three different, detection antibodies,the signals from SLT-1A-FR::scFv1 was set at 100%, and the relativesignals as a percentage of this control were determined for eachcell-targeting molecule sample tested by the calculation (Abs signal ofthe sample/Average Abs signal of the control)×100. The ELISA results areshown in FIG. 14.

FIG. 14 shows that, under native conditions, the exemplarycell-targeting molecules SLT-1A-combo7::scFv-1 (SEQ ID NO:44),SLT-1A-combo10::scFv-1 (SEQ ID NO:47), or SLT-1A-combo14::scFv-1 (SEQ IDNO:50) exhibit decreased antigenicities as compared to SLT-1A-FR::scFv-1(SEQ ID NO:34). These results demonstrate that SLT-1A-combo7,SLT-1A-combo10, and SLT-1A-combo14 exhibit decreased antigenicities ascompared to SLT-1A-FR (SEQ ID NO:5), or by inference or SLT-1A1-WT (SEQID NO:4), when linked by the same fashion to the same targeting domain(scFv-1) using the same linker. Additionally, the results shown in FIG.14 suggest that SLT-1A-combo7 and SLT-1A-combo14 have reduced relativeantigenicities as compared to SLT-1A-combo10 under the native conditionsof this ELISA assay.

F. Testing the CD4+ T-Cell De-Immunization of Exemplary, Shiga ToxinEffector Polypeptides SLT-1A-Combo(n)

Disruptions in predicted CD4+ T-cell epitope regions are tested forreductions in CD4+ T-cell immunogenicity using assays of human CD4+T-cell proliferation in the presence of exogenously administeredpolypeptides and assays of human CD4+ dendritic T-cell stimulation inthe presence of human monocytes treated with administered polypeptides.

T-cell proliferation assays known to the skilled worker are used to testthe effectiveness of CD4+ T-cell epitope de-immunization of Shiga toxineffector polypeptides SLT-1A-combo(n). The T-cell proliferation assay ofthis Example involves the labeling of CD4+ T-cells and then measuringchanges in proliferation using flow cytometric methods in response tothe administration of different peptides derived from either a Shigatoxin effector polypeptide combo(n) or a reference molecule, such as,e.g., a wild-type Shiga toxin A1 fragment, SLT-1A-FR, and/or a relatedcell-targeting molecule comprising the aforementioned.

A series of overlapping peptides derived from the chosen molecule aresynthesized and tested in the CFSE CD4+ T cell proliferation assay(ProImmune Inc., Sarasota, Fla., U.S). Human CD8+ T-cell depleted,peripheral blood mononuclear cells (PBMCs) labeled with CFSE arecultured with 5 μM of each peptide of interest for seven days in sixreplicate wells. Each assay plate includes a set of untreated controlwells. The assay also incorporates reference antigen controls,comprising synthetic peptides for known MHC class II antigens oragretopes.

The CD8+ T-cell depleted, PBMCs that proliferate in response to anadministered peptide will show a reduction in CFSE fluorescenceintensity as measured directly by flow cytometry. For a naïve T-cellanalysis, the Percentage Stimulation above background is determined foreach stimulated sample, through comparison with results from anunstimulated sample, such as by ranking with regard to fluorescentsignal, as negative, dim, or high. Counts for the CD4+ CFSE T-cell dimpopulation in each sample are expressed as a proportion of the totalCD4+ T-cell population. The replicate values are used to calculatePercentage Stimulation above Background (proportion of CD4+ T-cell CFSEdim cells with antigen stimulation, minus proportion of CD4+ T-cell CFSEdim cells without antigen stimulation). The mean and standard error ofthe mean are calculated from the replicate values. A result isconsidered “positive” if the Percentage Stimulation above background isgreater than 0.5% and also greater than twice the standard error abovebackground. To allow for comparison of peptides, a Response Index iscalculated. This index is based on multiplying the magnitude of response(Percentage Stimulation above background) for each peptide by the numberof responding donors (Percentage Antigenicity) for each peptide.

G. Determining the Relative, CD4+ T-Cell Immunogenicities of Exemplary,Shiga Toxin Effector Polypeptides SLT-1A-Combo(n)

The relative CD4+ T-cell immunogenicity of molecules of the invention isdetermined using the following dendritic cell (DC) T-cell proliferationassay. This DC T-cell assay measures CD4+ T-cell responses toexogenously administered polypeptides or proteins. The DC T-cell assayis performed using ProImmune's DC-T assay service to determine therelative levels of CD4+ T-cell driven immunogenicity between proteinsand cell-targeting molecules of the present invention as compared toreference molecules. The DC T-cell assay of this Example involvestesting human dendritic cells for antigen presentation of peptidesderived from the administered polypeptide, protein, or cell-targetingmolecule samples.

Briefly, healthy human donor tissues are used to isolate typed samplesbased on high-resolution MHC class II tissue-typing. A cohort of 20, 40or 50 donors is used. First, monocytes obtained from human donor PBMCsare cultured in a defined medium to generate immature dendritic cells.Then, the immature dendritic cells are stimulated with a well-definedcontrol antigen and induced into a more mature phenotype by furtherculture in a defined medium. Next, CD8+ T-cell depleted donor PBMCs fromthe same human donor sample are labeled with CFSE. The CFSE-labeled,CD8+ T-cell depleted PBMCs are then cultured with the antigen-primed,dendritic cells for seven days to allow for CD4+ dendritic cellstimulation, after which eight replicates for each sample are tested. Asnegative controls, each dendritic cell culture series also includes aset of untreated dendritic cells. For a positive control, the assayincorporates two well-defined reference antigens, each comprising afull-length protein.

To evaluate dendritic cell based immunogenicity, the frequency of donorcell responses is analyzed across the study cohort. Positive responsesin the assay are considered indicative of a potential in vivo CD4+T-cell response. A positive response, measured as a percentage ofstimulation above background, is defined as percentages greater than 0.5percent (%) in two or more independent donor samples. The strength ofpositive donor cell responses is determined by taking the meanpercentage stimulation above background obtained across accepted donorsfor each sample. A Response Index is calculated by multiplying the valueof the strength of response by the frequency of the donors responding todetermine levels of CD4+ T-cell immunogenicity for each sample. Inaddition, a Response index, representing the relative CD4+ T-cellimmunogenicity is determined by comparing the results from two samples,one involving a Shiga Toxin Effector Polypeptides SLT-1A-combo(n) and asecond variant which is a related molecule that lacks one or morepredicted disruptions of a CD4+ T-cell epitope and/or epitope region asa reference molecule.

H. Testing for Reductions in Immunogenicities of Exemplary,Cell-Targeting Molecules Comprising Shiga Toxin Effector PolypeptidesSLT-1A-Combo(n)

Mice were used to investigate the immunogenic potential of certainexemplary molecules of the present invention. The relativeimmunogenicities of exemplary cell-targeting molecules were determinedusing an assay for in vivo antibody responses to the cell-targetingmolecules after repeat, parenteral administrations over periods of manyweeks (see e.g. WO 2015/113005). An in-solution ELISA was used todetermine the relative amount of serum murine antibodies that werespecific to different cell-targeting molecules. This immunogenicityassay involves the use of mice which are indicative of the relativeimmunogenicities of molecules in mammals generally.

This assay was used to determine the relative immunogenicity ofexemplary cell-targeting molecules comprising SLT-1A-combo(n)::scFv-(n)as compared to the less de-immunized, cell-targeted moleculeSLT-1A-FR::scFv(n) or to the reference molecule scFv-3::SLT-1A-WT (SEQID NO:33). The reference molecule scFv-3::SLT-1A-WT was constructed inthe reverse, amino-carboxy fusion orientation of the exemplarycell-targeting molecules of the present invention tested in the relativeimmunogenicity assay, and the Shiga toxin effector polypeptide componentof scFv-3::SLT-1A-WT consisted of a wild-type Shiga toxin A1 fragment(SEQ ID NO:4), which represents an “un-de-immunized” Shiga toxineffector polypeptide.

Four different mouse studies were conducted where BALB/c or C57BL/6 micewere randomly assigned to treatment groups consisting of six mice pergroup and where the mice in different treatment groups were administereddifferent cell-targeting molecules. First, serum samples were collectedfrom each mouse prior to exposure to a cell-targeting molecule. Next,each mouse in a treatment group was administered 0.25 milligram of thesample molecule per kilogram of body weight (mg/kg) per dose of thesample cell-targeting molecule by intra-peritoneal injection three timesa week for two weeks. After a week without administration of anysamples, intra-peritoneal injections of 0.25 mg/kg per dose of thesample cell-targeting molecule were administered three times a week foran additional two weeks, resulting in a total of 12 doses ofcell-targeting molecule over a five-week interval. For all studies, theadministrated molecules SLT-1A-combo1::scFv-1(SEQ ID NO:43),SLT-1A-combo7::scFv-1(SEQ ID NO:44), SLT-1A-combo10::scFv-1(SEQ IDNO:47), SLT-1A-combo10::scFv-2 (SEQ ID NO:61), SLT-1A-combo12::scFv-1(SEQ ID NO:49), SLT-1A-combo15::scFv-1 (SEQ ID NO:51),SLT-1A-combo16::scFv-1 (SEQ ID NO:52), SLT-1A-combo19::scFv-1(SEQ IDNO:55), SLT-1A-combo22::scFv-2(SEQ ID NO:63), and the referencemolecules SLT-1A-FR::scFv-1 (SEQ ID NO:34) and SLT-1A-FR::scFv-2 (SEQ IDNO:35) were well-tolerated, resulting in no or only minimal effects onbody weight and no clinical signs. During and after the five-weekadministration interval, sera were collected from all the mice toobserve antibodies targeting the administered cell-targeting moleculesusing in-solution ELISAs. The mouse studies were done at Charles RiverLaboratories in Piedmont, N.C., U.S. The results of these studies areshown in Tables 13-16 and FIGS. 15-16.

The in-solution ELISAs to detect antibodies recognizing the administeredcell-targeting molecules were specific to the binding region scFv-(n) ofthe cell-targeting molecule being tested and performed as follows. Foreach cell-targeting molecule and its respective mouse-treatment group, adifferent ELISA assay was performed but using the same generalin-solution ELISA assay setup. For each cell-targeting molecule and itsrespective mouse-treatment group, the in-solution ELISA assay setupinvolved only the appropriate target biomolecule of the scFv-(n) of thecell-targeting molecule of that group. For all in-solution ELISA assays,the ELISA plate wells were coated with a target biomolecule of thecell-targeting, binding region scFv-(n). The same cell-targetingmolecule used for injections in a mouse-treatment group was incubatedovernight at 4° C. in solution with the serum collected from a singlemouse from that group, and then any complexes formed (e.g. complexescomprising the cell-targeting molecule and antibodies present in theserum) were captured using the coated ELISA plate wells. Captured,immune complexes comprising murine, immunoglobulin G molecules (IgGs)were detected using an anti-mouse IgG, secondary antibody conjugated tohorseradish peroxidase. HRP activity was detected in the wells by addinga chromogenic HRP substrate and then detecting light emission as aresult of chemiluminscence. The reaction was stopped by the addition ofHCl, and HRP activity or “ELISA signal” was measured as at 450 nM usinga plate reader. ELISA signal values were calculated as Absorbance values(Abs 450 nM) after subtracting the background signal as measured from“no serum” negative control wells. Serum was diluted to allow forAbsorbance value readings below the level of saturation for the assay,and the dilution ratio was the same for all mice in all treatment groupsmeasured on a given day. For the general setup of these in-solutionELISA assays, larger ELISA signal values indicate the presence of moremurine IgG antibodies recognizing the administered cell-targetingmolecule or in other words greater immunogenicity.

Based on these in-solution ELISA assays, none of the mice in anytreatment group of the four studies were observed to have pre-formedserum antibodies recognizing the cell-targeting molecules tested priorto exposure via injection. Thus, any post-administration detection ofanti-“cell-targeting molecule” IgG antibodies in the sera of the mice ofthese studies using the in-solution ELISA assay represents de novo,antibody production induced after the administration of a cell-targetingmolecule.

Murine IgG antibody responses to the administered cell-targetingmolecules were measured in the four studies at different time-pointsusing the appropriate in-solution ELISA assays, and the results arereported in Tables 13-16 and FIGS. 15-16. At each time-point, serumsamples were diluted such that the ELISA signal values remained withinthe dynamic range of the assay. For a single time-point in an individualstudy, all serum samples were diluted identically. The average ELISAAbsorbance values for each mouse-treatment group at individual serumcollection time-points (“average ELISA signal”) were calculated. Inconducting these relative immunogenicity studies, the samecell-targeting molecule injected into the mice of a particular treatmentgroup was used in the ELISA assay to capture sera antibodies from seracollected only from the mice of that same group. In other words,anti-“cell-targeting molecule” IgG antibodies present in the sera frommice administered SLT-1A-FR::scFv-1 (the SLT-1A-FR::scFv-1 referencegroup) were captured and detected in an ELISA assay designedspecifically and only with SLT-1A-FR::scFv-1, and, similarly,anti-“cell-targeting molecule” IgG antibodies present in the sera frommice administered SLT-1A-combo7::scFv-1 (e.g. the SLT-1A-combo7::scFv-1group) were captured and detected using an in-solution ELISA assaydesigned specifically and only with SLT-1A-combo7::scFv-1.

The average ELISA signal values for each mouse-treatment group atindividual serum collection time-points were calculated, and then therelative immunogenicity was calculated for each group at each time-pointrelative to the average ELISA Absorbance value at the same respectivetime-point for the reference group treated with the referencecell-targeting molecule as described above. The relative immunogenicityof each, tested, exemplary cell-targeting molecule(SLT-1A-combo(n)::scFv-(n)) in a given study at a certain time-point ascompared to the immunogenicity of a reference molecule (e.g.,SLT-1A-FR::scFv-(n) or scFv-3::SLT-1A-WT) was calculated using theformula: (ELISA signal of cell-targeting molecule−average ELISA signalof “no serum” control)/(average ELISA signal of referencemolecule−average ELISA signal of “no serum” control)×100. To createFIGS. 15-16, the percentage of the reference molecule ELISA signal foreach mouse-treatment group was graphed on the Y-axis, and the day ofserum collection was graphed on the X-axis. To measure mammalian IgGresponses to each cell-targeting molecule, the average ELISA signal(“avg signal”) and the percent of the avg signal of the referencemolecule SLT-1A-FR::scFv-(n) (“percent of ref”) were calculated for eachmouse-treatment group (Tables 13-16).

The results of the relative immunogenicity assays from the first mousestudy are presented in FIG. 15-panel A and Table 13. The term “N/A” wasused to indicate “not applicable” because calculations at Day 1 involvedpre-treatment serum with ELISA signal values of zero.

TABLE 13 Relative Immunogenicities of the Exemplary, Cell-TargetingMolecule SLT-1A-combo7::scFv-1 Compared to SLT-1A-FR::scFv-1 ReferenceDay of SLT-1A-combo7::scFv-1 SLT-1A-FR::scFv-1 serum avg ELISApercentage of avg ELISA percentage of collection signal reference signalreference 1 0 N/A 0 100 15 0.02 3.90% 0.49 100 22 0.10 11.20% 0.87 10029 0.20 27.30% 0.73 100 36 0.33 35.60% 0.93 100 43 0.20 24.60% 0.80 10050 0.19 26.30% 0.73 100

The results of the first, relative immunogenicity, mouse study were thatthe exemplary, cell-targeting molecule SLT-1A-combo7::scFv-1 exhibitedreduced immunogenicity as compared to the reference, cell-targetedmolecule SLT-1A-FR::scFv-1 at all time-points. The average ELISAAbsorbance values for the SLT-1A-combo7::scFv-1 treatment group werelower than for the SLT-1A-FR::scFv-1 treatment group at all time-pointsafter Day 1 (sera was collected pre-treatment on Day 1). For thecell-targeting molecules tested, anti-“cell-targeting molecule” IgGresponses were first observed on Day 15 (3 days after the administrationof the 6th dose), and, then, murine IgG responses were observed at allsubsequent time-points: Day 22 (10 days after the administration of the6th dose, prior to administration of the 7^(th) dose), Day 29 (3 daysafter the administration of the 9th dose prior to administration of the10^(th) dose), Day 36 (3 days after the administration of the lastdose), Day 43 (10 days after the administration of the 11^(th) dose),and Day 50 (17 days after the administration of the last dose) (FIG.15-panel A; Table 13). The data in Table 13 showed that thecell-targeting molecule SLT-1A-combo7::scFv-1 comprising thede-immunized, furin-cleavage resistant, Shiga toxin effector polypeptidecombo7 had reduced immunogenicity as compared to the cell-targetingmolecule SLT-1A-FR::scFv-1 comprising a wild-type Shiga toxin A1fragment. These results suggest that molecules comprising only Shigatoxin effector polypeptides that consist of the combination,de-immunized, protease cleavage-resistant, Shiga toxin effectorpolypeptide scaffold combo7 exhibit reduced immunogenicity compared tomolecules comprising a Shiga toxin effector polypeptide comprising (1) awild-type, Shiga-like toxin A1 fragment or (2) the furin-cleavageresistant, Shiga toxin effector polypeptide SLT-1A-FR.

The results of the relative immunogenicity assays from the second mousestudy are shown in FIG. 15-panel B and Table 14.

TABLE 14 Relative Immunogenicities of Exemplary, Cell-TargetingMolecules Comprising De-Immunized, Protease-Cleavage Resistant, ShigaToxin Effector Polypeptides Reference Day of SLT-1A-combo10::scFv-1SLT-1A-combo16::scFv-1 SLT-1A-combo19::scFv-1 SLT-1A-FR::scFv-1 serumavg percent avg percent avg percent avg collection signal of ref signalof ref signal of ref signal 1 0 N/A 0 N/A 0 N/A 0 15 0.36 19.2 0.26 13.51.21 63.7 1.9 22 0.44 24.3 0.44 24.4 1.29 71.2 1.81 29 0.27 19.5 0.7151.5 1.21 87.2 1.38 36 0.59 38.4 1.10 71.2 1.46 94.6 1.55 40 0.83 43.31.38 71.5 2.06 106.8 1.93

The results of the second, relative immunogenicity, mouse study werethat the exemplary, cell-targeting molecules tested(SLT-1A-combo10::scFv-1, SLT-1A-combo16::scFv-1, andSLT-1A-combo19::scFv-1) exhibited reduced immunogenicities as comparedto the reference cell-targeted molecule SLT-1A-FR::scFv-1 at alltime-points up to Day 36 (FIG. 15-panel B; Table 14). For thecell-targeting molecules tested, anti-“cell-targeting molecule” IgGresponses were first observed on Day 15 (3 days after the administrationof the 6th dose), and, then, murine IgG responses were observed at allsubsequent time-points: Day 22 (10 days after the administration of the6th dose, prior to administration of the 7^(th) dose), Day 29 (3 daysafter the administration of the 9th dose prior to administration of the10^(th) dose), Day 36 (3 days after the administration of the lastdose), and Day 40 (7 days after the administration of the last dose)(FIG. 15-panel B; Table 14). At both Day 15 and Day 22, the mice in thegroup administered the reference cell-targeted moleculeSLT-1A-FR::scFv-1 exhibited higher magnitudes of total IgG antibodyresponses as shown by the ELISA signal than the mice in the groupsadministered exemplary cell-targeting molecules comprising thede-immunized, protease-cleavage resistant, Shiga toxin effectorscaffolds (SLT-1A-combo10, SLT-1A-combo16, and SLT-1A-combo19) (FIG. 15;Table 14). At Days 29, 36, and 40, the percentages of the averagereference ELISA signal of the average ELISA signal values for the groupsadministered SLT-1A-combo16::scFv-1 and SLT-1A-combo19::scFv-1 werehigher than for the group administered SLT-1A-combo1::scFv-1. At alltime-points, the percentage of the average reference ELISA signal of theaverage ELISA signal values for the SLT-1A-combo19::scFv-1 group washigher than the percentage of the average ELISA signal values for thegroup administered SLT-1A-combo10::scFv-1 or SLT-1A-combo16::scFv-1.

The results of the relative immunogenicity assays from the third mousestudy are displayed in Table 15 and FIG. 15-panel C.

TABLE 15 Relative Immunogenicities of the Exemplary, Cell-TargetingMolecules Compared to Reference Molecules and Each Other reference 1reference 2 SLT-1A-combo10::scFv-2 scFv-3:: SLT-1A-FR percent of percentof SLT-1A-WT ::scFv-2 Day of serum avg ELISA ref 1 scFv- ref 2 SLT-1A-avg ELISA avg ELISA collection signal 3::SLT-1A-WT FR::scFv-2 signalsignal 1 0 N/A N/A 0 0 15 0.11 7.4% 4.9% 1.49 2.25 22 0.27 12.3% 9.6%2.20 2.81 29 1.28 40.6% 35.2% 3.15 3.64 SLT-1A-combo22::scFv-2 percentof percent of reference 1 reference 2 Day of serum avg ELISA ref 1 scFv-ref 2 SLT-1A- avg ELISA avg ELISA collection signal 3::SLT-1A FR::scFv-2signal signal 1 0 N/A N/A 0 0 15 0.60 40.3% 26.7% 1.49 2.25 22 1.1351.4% 40.2% 2.20 2.81 29 2.73 86.7% 75.0% 3.15 3.64 reference 3SLT-1A-combo10::scFv-2 SLT-1A-combo22:: percent of scFv-2 Day of serumavg ELISA ref 3 SLT-1A- avg ELISA collection signal combo22::scFv-2signal 1 0 N/A 0 15 0.11 18.3% 0.60 22 0.27 23.9% 1.13 29 1.28 46.9%2.73

The average ELISA Absorbance values for the SLT-1A-combo10::scFv-2 andSLT-1A-combo22::scFv-2 treatment groups were lower than for theSLT-1A-FR::scFv-2 and scFv-3:: SLT-1A-WT treatment groups at alltime-points after Day 1 up to Day 29. The data in Table 15 showed thatthe cell-targeting molecules SLT-1A-combo10::scFv-2 andSLT-1A-combo22::scFv-2, each comprising a de-immunized, furin-cleavageresistant, Shiga toxin effector polypeptide, exhibited reducedimmunogenicities compared to the cell-targeting moleculeSLT-1A-FR::scFv2 and the wildtype SLT-1 A1 fragment in the context of adifferent scFv (scFv-3) in the reverse, carboxy-amino fusionorientation. These results suggest that cell-targeting molecules whoseShiga toxin effector polypeptide region consists of the combination,de-immunized, protease cleavage-resistant, Shiga toxin effectorpolypeptide scaffold combo10 or combo22 show reduced immunogenicitiescompared to cell-targeting molecules whose Shiga toxin effectorpolypeptide region consists of (1) a wild-type, Shiga toxin effectorpolypeptide or (2) the furin-cleavage resistant, Shiga toxin effectorpolypeptide SLT-1A-FR.

The results of the relative immunogenicity assays from the fourth mousestudy are shown in FIG. 16 and Table 16.

TABLE 16 Relative Immunogenicities of Exemplary, Cell-TargetingMolecules as Compared to SLT-1A-FR::scFv-1 Reference 1 SLT-1A- SLT-1A-SLT-1A- SLT-1A- SLT-1A- Day of combo1::scFv-1 combo10::scFv-1combo12::scFv-1 combo15::scFv-1 FR::scFv-1 serum avg percent avg percentavg percent avg percent avg collection signal of ref 1 signal of ref 1signal of ref 1 signal of ref 1 signal 1 0 N/A 0 N/A 0 N/A 0 N/A 0 150.136 41.14 0.004 1.17 0.020 6.09 0.009 2.88 0.329 22 0.442 61.54 0.0233.24 0.063 8.71 0.053 7.43 0.719 29 0.937 47.81 0.065 3.33 0.547 27.890.215 10.98 1.959 36 1.949 94.08 0.551 26.60 1.117 53.90 0.551 26.592.072 40 2.247 99.19 0.873 38.55 1.576 69.57 0.832 36.75 2.265SLT-1A-combo10::scFv-1 Day of avg percent of ref Reference 2 serum ELISA2 SLT-1A- SLT-1A-combo1::scFv-1 collection signal combo1::scFv-1 avgELISA signal 1 0 N/A 0 15 0.004 2.85% 0.136 22 0.023 5.27% 0.442 290.065 6.97% 0.937 36 0.551 28.27% 1.949 40 0.873 38.87% 2.247

The results of the fourth immunogenicity study were that the exemplary,cell-targeting molecules tested exhibited reduced immunogenicities ascompared to the reference cell-targeting molecule SLT-1A-FR::scFv-1, atleast at earlier time-points (FIG. 14-panel A; Table 16). Sera wascollected pre-treatment on Day 1. For the cell-targeting moleculestested, anti-“cell-targeting molecule” IgG responses were first observedon Day 15 (3 days after the administration of the 6th dose), and, then,murine IgG responses were observed at all subsequent time-points: Day 22(10 days after the administration of the 6th dose, prior toadministration of the 7^(th) dose), Day 29 (3 days after theadministration of the 9th dose prior to administration of the 10^(th)dose), Day 36 (3 days after the administration of the last dose), andDay 40 (7 days after the administration of the last dose) (FIG. 14;Table 16). At both Day 15, Day 22 and Day 29, the mice in the groupadministered the reference cell-targeting molecule SLT-1A-FR::scFv-1exhibited higher magnitudes of total IgG antibody responses as shown bythe ELISA signal than the mice in the groups administered exemplarycell-targeting molecules comprising the de-immunized, protease-cleavageresistant, Shiga toxin effector scaffolds (SLT-1A-combo1,SLT-1A-combo10, SLT-1A-combo12, and SLT-1A-combo15) (FIG. 16; Table 16).At all time-points, the percentage of the average reference ELISA signalof the average ELISA signal values for the SLT-1A-combo1::scFv-1treatment group was higher than the percentage of the average ELISAsignal values for the treatment groups administeredSLT-1A-combo10::scFv-1, SLT-1A-combo12::scFv-1, andSLT-1A-combo15::scFv-1. At Days 36 and 40, the percentages of theaverage reference ELISA signal of the average ELISA signal values forthe treatment group administered SLT-1A-combo12::scFv-1 were higher thanfor the treatment group administered SLT-1A-combo10::scFv-1 andSLT-1A-combo15::scFv-1. These results suggest that cell-targetingmolecules whose Shiga toxin effector polypeptide region consists of thecombination, de-immunized, protease cleavage-resistant, Shiga toxineffector polypeptide scaffold SLT-1A-combo1, SLT-1A-combo10,SLT-1A-combo12, and SLT-1A-combo15 show reduced immunogenicity comparedto cell-targeting molecules whose Shiga toxin effector polypeptideregion consists of (1) a wild-type, Shiga toxin effector polypeptide or(2) the furin-cleavage resistant, Shiga toxin effector polypeptideSLT-1A-FR.

Of the cell-targeting molecules tested in the second, third, and fourthmouse studies, the combination, de-immunized, protease-cleavageresistant, Shiga toxin effector scaffold SLT-1A-combo10 appeared to bethe most de-immunized by this relative immunogenicity assay under theconditions tested (see FIGS. 15-16; Tables 14-16). This Shiga toxineffector scaffold comprised (1) five, disrupted epitope regions, two ofwhich involved multiple amino acid residue substitutions, and (2) anendogenous epitope region disrupted by an embedded, heterologous, T-cellepitope. In the second study, the combination, de-immunized,protease-cleavage resistant, Shiga toxin effector scaffoldSLT-1A-combo16 appeared to be more de-immunized than the combination,de-immunized, protease-cleavage resistant, Shiga toxin effector scaffoldSLT-1A-combo19, especially at earlier time-points (see FIG. 15-panel B;Table 14). In the fourth study, the combination, de-immunized,protease-cleavage resistant, Shiga toxin effector scaffold SLT-1A-combo1appeared to be the least de-immunized of the tested, Shiga toxineffector polypeptides SLT-1A-combos (1, 10, 12 and 15) (see FIG.16-panel A; Table 16). In the fourth study, the combination,de-immunized, protease-cleavage resistant, Shiga toxin effector scaffoldSLT-1A-combo15 appeared to be more de-immunized than SLT-1A-combo12,especially at later time-points (see FIG. 16-panel A; Table 16).

The results of these immunogenicity studies indicate that two types ofepitope region disruptions, amino acid residue substitutions andembedded, heterologous, CD8+ T-cell epitopes, can contribute to thede-immunization of a Shiga toxin effector polypeptide. Further, thecombination of both types of disruptions in the same Shiga toxineffector polypeptide can result in a more de-immunized, Shiga toxineffector polypeptide. The overall magnitude of antibody induction in themice in the groups administered exemplary, cytotoxic, cell-targetingmolecules comprising these combination, de-immunized, CD8+ T-cellhyper-immunized, protease-cleavage resistant, Shiga toxin effectorpolypeptides was reduced as compared to magnitude of antibody inductionin the mice in the group administered the cell-targeting moleculecomprising the protease-cleavage resistant but otherwise wild-type Shigatoxin effector polypeptide region. The decreases in the ELISA signalvalues of the cell-targeting molecules comprising certain, combination,de-immunized, protease-cleavage resistant, Shiga toxin effectorscaffolds demonstrates that these particular scaffolds were successfullyde-immunized (i.e. had reduced immunogenic potential in mammals). Thus,the Shiga toxin effector polypeptides SLT-1A-combo1, SLT-1A-combo7,SLT-1A-combo10, SLT-1A-combo12, SLT-1A-combo15, SLT-1A-combo16, andSLT-1A-combo19 exhibited reduced immunogenic potential in mammals, andany, exemplary, cell-targeting molecule comprising these polypeptidesshould be de-immunized as compared to an analogous molecule comprisingonly wild-type or merely protease-cleavage resistant, Shiga toxineffector polypeptides due to furin-cleavage motif mutations in thenatively positioned region from amino acid residues 238 to 257 in StxAand SLT-1A.

Differences observed in the immunogenic potentials of the differentcombination, de-immunized, protease-cleavage resistant, Shiga toxineffector polypeptides tested shows that certain combinations ofde-immunized sub-regions and/or embedded T-cell epitope sub-regionsreduce immunogenicity by different magnitudes. For example in study 2,SLT-1A-combo10 was more de-immunized than SLT-1A-combo16 andSLT-1A-combo 19, especially at later time-points (Table 14; FIG.15-panel B), despite combo16 and combo19 comprising more disruptedepitope regions and total amino acid residue substitutions compared to awild-type Shiga toxin A Subunit than SLT-1A-combo10 comprises (Table 8).In study 4, SLT-1A-combo10 was more de-immunized than SLT-1A-combo12,especially at later time-points (Table 16; FIG. 16-panel A) despite thatboth SLT-1A-combo12 and SLT-1A-combo15 comprise more disrupted epitoperegions and total amino acid residue substitutions compared to awild-type Shiga toxin A Subunit than SLT-1A-combo10 comprises (Table 8).Thus, the cumulative combination of additional B-cell epitope regiondisruptions did not necessarily result in additional decreases inimmunogenicity but rather may result in increases in unwantedimmunogenicities, such as, e.g., at later time-points (see FIG. 15-panelB and compare the results for SLT-1A-combo10 with the results forSLT-1A-combo16 and SLT-1A-combo19).

I. Testing the Ability of Exemplary, Cell-Targeting Molecules to Delivera T-Cell Epitope-Peptide to the MHC Class I Pathway of a Cell forPresentation

The presentation of a T-cell epitope by the MHC class I system targetsthe presenting cell for killing by CTL-mediated lysis and also triggersimmune stimulation in the local area. By engineering cell-targetingmolecules comprising Shiga toxin effector polypeptides comprisingheterologous, immunogenic epitopes, the targeted delivery andpresentation of immuno-stimulatory antigens may be accomplished. Thepresentation of immuno-stimulatory non-self antigens, such as e.g. knownviral antigens with high immunogenicity, by target cells signals toother immune cells to destroy the target cells as well as to recruitmore immune cells to that area within an organism.

In order to simultaneously de-immunize and provide for T-cell epitopepresentation on the target cell surface within the same Shiga toxineffector polypeptide region, a predicted B-cell epitope region wasdisrupted by replacing amino acid residues within it with an immunogenicT-cell epitope region predicted to bind to human MHC class I molecules.

In this Example, the abilities of exemplary cell-targeting molecules ofthe present invention to deliver T-cell epitopes to the MHC class Ipathway of target cells for presentation to the target cell surface areinvestigated. In addition, the functional consequences of target cells'MHC class I presentation of T-cell epitopes delivered by exemplarycell-targeting molecules of the present invention are investigated byobserving various immune responses induced by the presentation of thedelivered epitope-peptide by an MHC I molecule.

1. Testing the Ability of a Molecule to Deliver a T-Cell Epitope-Peptideto the MHC Class I Pathway for Presentation on the Cell Surface

Routine assays known in the art are used to investigate the ability ofexemplary molecules of the present invention to deliver a T-cell epitopeto a MHC class I molecule (see e.g. WO 2015/113007). In particular, aflow cytometry method is used to demonstrate delivery and extracellulardisplay of a T-cell epitope-peptide (inserted or embedded in a Shigatoxin effector polypeptide) in complex with MHC class I molecules on thesurfaces of target cells. This flow cytometry method utilizes solublehuman T-cell receptor (TCR) multimer reagents (Soluble T-Cell AntigenReceptor STAR™ Multimer, Altor Bioscience Corp., Miramar, Fla., U.S.),each with high-affinity binding to a different epitope-human HLAcomplex.

Each STAR™ TCR multimer reagent is derived from a specific T-cellreceptor and allows detection of a specific peptide-MHC complex based onthe ability of the chosen TCR to recognize a specific peptide presentedin the context of a particular MHC class I molecule. These TCR multimersare composed of recombinant human TCRs which have been biotinylated andmultimerized with streptavidin. The TCR multimers are labeled withphycoerythrin (PE). These TCR multimer reagents allow the detection ofspecific peptide-MHC class I complexes presented on the surfaces ofhuman cells because each soluble TCR multimer type recognizes and stablybinds to a specific peptide-MHC complex under varied conditions (Zhu Xet al., J Immunol 176: 3223-32 (2006)). These TCR multimer reagentsallow the identification and quantitation by flow cytometry ofpeptide-MHC class I complexes present on the surfaces of cells.

The target cells used in this Example are available from the ATCC(Manassas Va., U.S.), National Cancer Institute of the U.S. (Frederick,Md., U.S.), and/or DSZM (Braunschweig, Del.). Using standard flowcytometry methods known in the art, the target cells are confirmed toexpress on their cell surfaces both the appropriate MHC-class I moleculeand the extracellular target biomolecule of the cell-targeting moiety ofthe cell-targeting molecules used in this Example.

The target cells are treated with exemplary cell-targeting molecules ofthe present invention that each comprise a Shiga toxin effectorpolypeptide comprising an embedded or inserted T-cell epitope. Certainexemplary molecules of the present invention tested in this Example arecatalytically impaired or inactivated by the addition of one or both ofthe following mutations: Y77S and E167D. Sets of target cells aretreated by exogenous administration of the different exemplarycell-targeting molecules of the invention at concentrations similar tothose used by others taking into account cell-type specificsensitivities to Shiga toxins (see e.g. Noakes K et al., FEBS Lett 453:95-9 (1999)). The treated cells are then incubated for six hours instandard conditions, including at 37° C. and an atmosphere with 5%carbon dioxide, to allow for intoxication mediated by a Shiga toxineffector polypeptide region. Then the cells are washed with cell culturemedium, re-suspended in fresh cell culture medium, and incubated for 20hours prior to staining with the appropriate STAR™ multimer reagent.Additional time-points and setup conditions are also tested.

As controls, sets of target cells are treated in three conditions: 1)without any treatment (“untreated”) meaning that no exogenous moleculesare added, 2) with exogenously administered control antigen-peptide, and3) with exogenously administered control antigen-peptide combined with aPeptide Loading Enhancer (“PLE,” Altor Bioscience Corp., Miramar, Fla.,U.S.). The control antigen-peptide peptide combined with PLE treatmentallowed for exogenous peptide loading and served as a positive control.Cells displaying the appropriate MHC class I haplotype can be forced toload the appropriate exogenously applied peptide from an extracellularspace (i.e. in the absence of cellular internalization of the appliedpeptide) or in the presence of PLE, which is a mixture ofB2-microglobulin and other components.

After the treatments, all the sets of cells are washed and incubatedwith the appropriate STAR multimer reagent for one hour on ice. Thecells are washed and the fluorescence of the samples are measured byflow cytometry using an Accuri™ C6 flow cytometer (BD Biosciences, SanJose, Calif., U.S.) to detect the presence of and quantify any STAR™multimer bound to cells in the population (sometimes referred to hereinas “staining”).

The untreated control is used to identify the positive and negative cellpopulations by employing a gate which results in less than 1% of cellsfrom the untreated control in the “positive” gate (representingbackground signal). The same gate is then applied to the other samplesto characterize the positive population for each sample.

The detection of the exogenously administered, embedded or insertedT-cell epitope complexed with human MHC class I molecules on the cellsurface of intoxicated target cells demonstrates that cell-targetingmolecules comprising the embedded or inserted T-cell epitope-peptide arecapable of entering target cells, performing sufficient sub-cellularrouting, and delivering enough T-cell epitope to the MHC class I pathwayfor surface presentation on the target cell surface. 2. Testing theAbility of a Molecule to Induce Cytotoxic T-Cell Mediated Cytolysis ofTarget Cells and Other Immune Responses

Routine assays known in the art are used to investigate the functionalconsequences of target cells' MHC class I presentation of T-cellepitopes delivered by exemplary cell-targeting molecules of theinvention (see e.g. WO 2015/113007). The functional consequences toinvestigate include CTL activation, CTL-mediated target-cell-killing,and cytokine release by CTLs.

A CTL-based cytotoxicity assay is used to assess the consequences ofepitope presentation. The assay involves tissue-cultured target cellsand T-cells. Target cells are intoxicated as described in WO2015/113007. Briefly, target cells are incubated for six hours instandard conditions with different exogenously administered,cell-targeting molecules, where certain cell-targeting moleculescomprise a Shiga toxin effector polypeptide of the invention. Next, CTLsare added to the intoxicated target cells and incubated to allow for theT-cells to recognize and bind any target-cells displayingepitope-peptide/MHC class I complexes. Then certain functionalconsequences are investigated using standard methods known to theskilled worker, including CTL binding to target cells,target-cell-killing by CTL-mediated cytolysis, and the release ofcytokines, such as interferon gamma or interleukins by ELISA or ELIspot.

The activation of CTLs by target cells displaying epitope-peptide/MHCclass I complexes is quantified using commercially available CTLresponse assays, e.g. CytoTox96® non-radioactive assays (Promega Corp.,Madison, Wis., U.S.), Granzyme B ELISpot assays (Mabtech, Inc.,Cincinnati, Ohio, U.S.), caspase activity assays, and LAMP-1translocation flow cytometric assays. To specifically monitorCTL-mediated killing of target cells, carboxyfluorescein succinimidylester (CFSE) is used to target-cells for in vitro and in vivoinvestigation as described in the art (see e.g. Durward M et al., J VisExp 45 pii 2250 (2010)).

Summary of Example 2

Exemplary, cell-targeting molecules comprising combination, Shiga toxineffector polypeptide with multiple B-cell epitope region disruptionswere de-immunized as shown by reductions in both antigenicity andimmunogenicity as compared to reference molecules. In addition, thisExample shows that certain, de-immunized cell-targeting moleculescomprising combination, Shiga toxin effector polypeptides comprisingmultiple B-cell epitope region disruptions, furin cleavage motifdisruptions, and/or embedded T-cell epitopes retain at a significantlevel of one or more Shiga toxin effector functions, such as, e.g.,catalytic ribosome inhibition, intracellular routing, and cytotoxicity.

Table 17 summarizes results from Example 2 for the exemplary,protease-cleavage resistant, de-immunized, cell-targeting molecules ofthe present invention which comprise the Shiga toxin effectorpolypeptide SLT-1A-combo1, SLT-1A-combo7, SLT-1A-combo10,SLT-1A-combo12, SLT-1A-combo15, SLT-1A-combo16, or SLT-1A-combo19.

TABLE 17 Summary of Exemplary Cell-Targeting Molecules EmpiricallyTested for Cytotoxicity and Reduced Immunogenic Potential in MammalsCytotoxicity Immunogenicity cell-targeting (CD₅₀ in nM) compared to SLT-molecule scaffold Varies with the cell-line 1A-FR:scFv-1 SLT-1A- 0.0241-99%  combol::scFv-1 SLT-1A- 0.01, 0.02, 0.03, 0.12, 0.13, 4-36%combo7::scFv-1 0.39 SLT-1A- 0.03, 0.07, 0.08, 0.25, 0.84 1-43%combo10::scFv-1 SLT-1A- 0.07, 0.18 6-70% combo12::scFv-1 SLT-1A- 0.05,0.09 3-37% combo15::scFv-1 SLT-1A- 0.03, 0.12 14-72%  combo16::scFv-1SLT-1A- 0.03, 0.16 64-107%  combo19::scFv-1 SLT-1A-FR 0.01, 0.02, 0.05,0.10, 0.28  100% ::scFv-1

The exemplary, protease-cleavage resistant, de-immunized, cell-targetingmolecules of the invention shown in Table 17 all exhibited significantlevels of cytotoxicity which were comparable to cell-targeting moleculescomprising wild-type Shiga toxin A1 fragments. These exemplary,protease-cleavage resistant, de-immunized, cell-targeting molecules ofthe invention exhibit catalytic inhibition of translation by ribosomes,intracellular routing comparable to wild-type a Shiga toxin A Subunitand/or A1 fragment, and cytotoxicity comparable to a cell-targetingmolecules comprising a wild-type Shiga toxin A1 fragment and/or ASubunit. These exemplary, protease-cleavage resistant, de-immunized,cell-targeting molecules of the invention exhibit levels of catalyticactivity comparable to a wild-type, Shiga toxin A Subunit and/or A1fragment. These exemplary, protease-cleavage resistant, de-immunized,cell-targeting molecules of the invention exhibit levels of cytotoxicitycomparable to a wild-type Shiga toxin A Subunit and/or A1 fragment.These cell-targeting molecules of the invention all exhibited reducedimmunogenicity in mammals as compared to cell-targeting moleculescomprising a Shiga toxin effector polypeptide region consisting of (1) awild-type, Shiga toxin A1 fragment and/or (2) the furin-cleavageresistant, Shiga toxin effector polypeptide SLT-1A-FR. Furthermore,certain cell-targeting molecules of the present invention exhibitincreased stability, improved in vivo tolerability, and/or the abilityto deliver heterologous, T-cell epitopes for MHC class I presentation bytarget cells.

The results shown in Example 2 reinforce the idea that various,exemplary, epitope region disruptions may be combined together in asingle molecule to create greater reductions in antigenicity and/orimmunogenicity while still retaining significant levels of one or moreShiga toxin effector function (see e.g. WO 2015/113005) and sometimesproviding another functional feature(s) not present in wild-type Shigatoxin A Subunits, such as, e.g., furin-cleavage resistance and/or theability to deliver a heterologous, T-cell epitope to a target cell.Certain, combination, de-immunized, Shiga toxin A Subunit effectorpolypeptides of the present invention exhibit synergistic reductions inimmunogenicity as compared to the sum of their partially de-immunizedsub-regions, such as, e.g., SLT-1A-combo7 SLT-1A-combo10, andSLT-1A-combo15.

The following substitutions have been made and tested in at least oneShiga toxin effector polypeptide which retained a significant level ofin vitro, ribosome inhibition and/or cytotoxicity: K1A, K1M, T4I, S81,T9I, K11A, S33I, S33C, S43N, G44L, S45V, T45V, S45I, T45I, G46P, D47M,N48V, L49A, F50T, A51V, D53A, D53N, V54L, V54I, R55A, R55V, R55L, G56P,I57F, I57M, D58A, D58V, D58F, P59A, P59F, E601, E60T, E60R, E61A, E61V,E61L, G62A, D94A, S96I, T104N, A105L, T107P, L108M, S109V, G110A, D111T,D141A, V154A, G147A, T180G, T1811, D183A, D183G, D184A, D184A, D184F,L185D, S186A, S186F, G187A, G187T, R188A, S189A, D198A, R205A, C242S,R248A, and R251A.

Despite the challenges predicting successful substitutions apriori, thedata provided in the Examples herein give reasons to believe thatcertain amino acid substitutions are likely to successfully reduceantigenicity and/or immunogenicity while maintaining significant Shigatoxin effector function(s). The term “successful” is used here to meanone or more amino acid residue substitutions in a predicted epitoperegion resulted in a Shiga toxin effector polypeptide which retained oneor more Shiga toxin effector functions. For example, substitutions atspecific amino acid positions shown herein as successfully toleratingsubstitutions are likely to be successful for retaining at least oneShiga toxin effector function when substituted with certain other aminoacids. Successful single amino acid substitution may generally becombined with other successful amino acid substitutions in a differentepitope region to generate de-immunized, Shiga toxin effectorpolypeptides which retain significant Shiga toxin effector function(s).Similarly, the demonstration that cell-targeting molecules comprisingShiga toxin effector polypeptide regions with multiple single amino acidsubstitutions within the same epitope region retained enzymatic activitysuggests that successful single amino acid substitution in the sameepitope region may generally be combined with other single amino acidsubstitutions in the same epitope region to generate de-immunized, Shigatoxin effector polypeptides which retain significant Shiga toxineffector function(s).

It has been empirically demonstrated that certain substitutions (K1A,K1M, T4I, S81, T9I, K11A, S33I, S33C, S43N, G44L, S45V, T45V, S45I,T45I, G46P, D47M, N48V, L49A, F50T, A51V, D53A, D53N, V54L, V54I, R55A,R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F, P59A, P59F, E601, E60T,E60R, E61A, E61V, E61L, G62A, D94A, S96I, T104N, A105L, T107P, L108M,S109V, G110A, D111T, D141A, G147A, V154A, G147A, T180G, T1811, D183A,D183G, D184A, D184A, D184F, L185D, S186A, S186F, G187A, G187T, R188A,S189A, D198A, R205A, C242S, R248A, R251A, and/or combinations thereof)and certain positions tolerated substitutions (1, 4, 8, 9, 11, 33, 43,44, 45, 46, 47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,94, 96, 104, 105, 107, 108, 109, 110, 111, 141, 147, 154, 180, 181, 183,184, 185, 186, 187, 188, 189, 198, 205, 242, 248, and 251) whileretaining a significant level of activity for at least one Shiga toxineffector function. This empirical data suggest certain other epitopedisrupting substitutions and combinations of epitope disruptingsubstitutions which may be used to generate de-immunized, Shiga toxineffector polypeptides which retain significant Shiga toxin effectorfunction(s). It is predictable that other amino acid substitutions toamino acid residues of a conservative functional group will also betolerated. For example, other substitutions known to the skilled workerto be similar to any of K1A, K1M, T4I, S81, T9I, K11A, S33I, S33C, S43N,G44L, S45V, T45V, S45I, T45I, G46P, D47M, N48V, L49A, F50T, A51V, D53A,D53N, V54L, V54I, R55A, R55V, R55L, G56P, I57F, I57M, D58A, D58V, D58F,P59A, P59F, E601, E60T, E60R, E61A, E61V, E61L, G62A, D94A, S96I, T104N,A105L, T107P, L108M, S109V, G110A, D111T, D141A, G147A, V154A, G147A,T180G, T1811, D183A, D183G, D184A, D184A, D184F, L185D, S186A, S186F,G187A, G187T, R188A, S189A, D198A, R205A, C242S, R248A, or R251A willalso be able to disrupt an epitope while maintaining at least one Shigatoxin effector function.

Example 3. Furin-Cleavage Resistant, Shiga Toxin a Subunit EffectorPolypeptides and Cell-Targeting Molecules Comprising the Same

Furin-cleavage resistant, Shiga toxin A Subunit effector polypeptideswere created and tested as components of cell-targeting moleculeswherein each cell-targeting molecule comprised a cell-targeting,immunoglobulin-type, binding region. To engineer protease resistanceinto a Shiga toxin effector polypeptide, two amino acid residuesubstitutions, R248A and R251A, were introduced into Shiga toxineffector polypeptides as described in Example 2. The Shiga toxineffector polypeptide SLT-1A-FR (SEQ ID NO:5) was used to create thecell-targeting molecule SLT-1A-FR::scFv-9 (SEQ ID NO:41).

A. Quantifying Furin Cleavage of a Molecule of the Present InventionRelative to a Reference Molecule

The cell-targeting molecule SLT-1A-FR::scFv-9 (SEQ ID NO:41) was testedusing an in vitro, furin-cleavage assay to quantify furin cleavage ascompared to a wild-type control using methods known to the skilledworker (see e.g. WO 2015/191764). To assess the ability of furin tocleave SLT-1A-FR::scFv-9, purified protein samples in phosphate bufferedsaline (PBS) were incubated with furin (New England Biolabs, Ipswich,Mass., U.S.) at 0.5 furin activity units (U) per microgram (pg) ofsample protein in furin cleavage buffer (100 millimolar (mM) HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7, 1 mM CaCl₂)for 30 hours at 30° C. Control samples were incubated without furin at4° C. or 30° C. in the same buffer. The various, cell-targeting moleculesamples were electrophoresed on SDS, polyacrylamide gels underdenaturing conditions and stained with Coomassie (FIG. 17).

FIG. 17 shows a picture of a gel with the lanes numbered and a figurelegend indicating which lane was loaded with which sample: either acell-targeting molecule comprising a wild-type Shiga toxin effectorpolypeptide region (SLT-1A-WT) or a furin-cleavage site disrupted, Shigatoxin effector polypeptide (SLT-1A-FR). The lanes marked “MW Marker”show the migration pattern of a protein molecular weight ladder alongwith the approximate size of individual ladder protein bands inkiloDaltons (kDa) for use as an internal molecular weight reference thatallows for the estimation of the sizes of proteins in the numberedlanes. The figure legend indicates the pre-treatment conditions of thecell-targeting molecule samples with the temperature in degrees Celsius(° C.), duration, and whether any furin was added by denoting the amountof furin activity units per microgram (labeled “U/pg furin”) or “nofurin” for zero units.

FIG. 17 shows that SLT-1A-FR::scFv-9 (SEQ ID NO:41) was resistant toproteolytic cleavage by human furin. The cell-targeting molecules testedin this assay were both about 56 kDa in size and comprised a Shiga toxineffector polypeptide of about 28 kDa (identical in size for bothSLT-1A-WT or SLT-1A-FR) linked to a carboxy-terminal linker and bindingregion which together were about 28 kDa in size. If furin cleavage hadoccurred in the surface exposed, extended loop 242-251 of SLT-1A, thenthe expected result would be two protein bands with near equal molecularweights of around 28 kDa each. If furin cleavage occurs precisely at thecarboxy peptide bond of the arginine at position 251 of the WT scaffoldin SLT-1A-WT::scFv-9, then the two resulting protein bands should havethe molecular weights of 27.5 kDa for SLT-1A (either WT or FR) and of28.3 kDa for scFv-9.

The gel was analyzed using Gel Analyzer 2010 software. This softwaredetected the lanes of the gel pictured in FIG. 17 and the bands withineach lane. Using background subtraction mode, the background wasautomatically defined and subtracted from the volumes of all bandsanalyzed using a rolling ball background subtraction with the ballradius set at 25. The results of this quantitative band analysis of thegel pictured in FIG. 17 is summarized in Table 18. Table 18 shows therelative mobility and raw volume values for certain bands migratingaround 56 kDa in lanes 1-6 in the gel pictured in FIG. 17. The “nofurin” 4° C. treatment of the cell-targeted molecule (SLT-1A-WT::scFv-9)(SEQ ID NO:42), which comprises a wild-type Shiga toxin effectorpolypeptide, results in lane #2 was used as a control to determine thepercentage of uncleaved material at 30° C. in the absence or presence offurin (lanes #2 and #3, respectively). For the SLT-1A-FR::scFv-9samples, the “no furin” 4° C. lane (lane #4) was used to determine thepercentage of uncleaved material at 30° C. in the absence or presence offurin (lanes #5 and #6, respectively). For each molecule, the“percentage uncleaved” of the ˜56 kDa band for each furin treated samplewas calculated by the following formula: (Raw Volume of ˜56 kDa sampleband)/(Raw Volume of ˜56 kDa band from the “no furin” 4° C.treatment)×100 (see Table 18).

TABLE 18 Quantification of Furin-Cleavage Relative to a ReferenceMolecule Relative mobility (R_(f)) Raw Percentage Treatment Lane # of~56 kDa Band Volume uncleaved SLT-1A-WT::scFv-9 no furin, 4° C. 1 0.39753 100.0% no furin, 30° C. 2 0.36 766 101.7% 0.5 U/μg furin, 3 0.34 24632.7% 30° C. SLT-1A-FR::scFv-9 no furin, 4° C. 4 0.37 995 100.0% nofurin, 30° C. 5 0.39 985 99.0% 0.5 U/μg furin, 6 0.37 1000 100.5 30° C.

For the wild-type furin site samples, the amount of protein in the ˜56kDa band for the sample incubated with furin was reduced compared to theamount of protein in the ˜56 kDa band for “no furin” samples (Table 18;FIG. 17, lane #3 compared to lanes #1 and #2). The furin treatment ofSLT-1A-WT::scFv-9 resulted in the production of two new bands of about28 kDa (FIG. 17, lane #3), which match the expected sizes offurin-cleavage products resulting from cleavage at the carboxy-terminusof the Shia toxin A1 fragment region of SLT-1A-WT::scFv-9. For theSLT-1A-FR::scFv-9 samples, the amount of protein in the ˜55 kDa band inthe sample incubated with furin lane appeared unchanged from the amountof protein in the ˜55 kDa band for “no furin” samples (Table 18; FIG.17, lane #6 compared to lanes #4 and #5).

This quantitative analysis showed that a cell-targeting moleculedesigned with the wild-type Shiga toxin effector polypeptide SLT-1A-WTand a carboxy-terminal binding region exhibited about 67.3% cleavagewith about 32.7% of SLT-1A-WT::scFv-9 remaining uncleaved. Thepercentage of furin-cleavage as compared to a reference molecule can beexpressed as a ratio of [(available material—uncleaved)/availablematerial] of the molecule of interest to [(available material—uncleaved)/available material] of the reference molecule. In thisassay, SLT-1A-FR::scFv-9 exhibited[(985-1000)/1000]/[(766-246)/766]=−1.5% cleavage of the reference orapproximately zero cleavage.

This assay showed that a cell-targeting molecule designed with the Shigatoxin effector polypeptide SLT-1A-FR exhibited 0% cleavage with 100% ofthe cell-targeting molecule remaining uncleaved. Thus, the SLT-1A-FRscaffold appears to be resistant to furin cleavage in this assay underthe conditions tested.

B. Testing the In Vivo Tolerability of Cell-Targeting Molecules of thePresent Invention Using Laboratory Animals

The in vivo tolerability of exemplary, cell-targeting molecules of thepresent invention is tested using mice in order to determine the degreeto which overt adverse effects were detected at various dosages ofcell-targeting molecule samples. The tolerability studies are performedusing methods known to the skilled worker and/or described herein (seee.g. WO 2015/191764). For example, mice are injected with cell-targetingmolecule samples or vehicle controls at doses ranging from 0.25 to 5.00milligrams per kilogram of body weight per injection (mg/kg/inj) forthree times a week over several weeks. In order to assess in vivotolerability, the injected mice are monitored for changes in health andclinical signs, such as, e.g., aspects of morbidity, morbundity, bodyweight, physical appearance, measureable clinical signs, unprovokedbehavior, and responses to external stimuli, such as, (see e.g. MortonD, Griffiths P, Vet Rec 116: 431-43 (1985); Montgomery C, Cancer Bull42: 230-7 (1990); Ullman-Culleré M, Foltz C, Lab Anim Sc 49: 319-23(1999); Clingerman K, Summers L, J Am Assoc Lab Anim Sci 51: 31-6(2012)). Euthanasia may be used in response to signs of morbidity and/ormorbundity and, thus, create a mortality time-point. For example, adecrease in body weight of 15-20% in 2-3 days can be used as a sign ofmorbidity in rodents and as a justification for euthanization (see e.g.Institute of Laboratory Animal Research 2011. Guide for the care and useof laboratory animals, 8th ed., Washington, D.C., U.S.: NationalAcademies Press).

The cell-targeting molecules of the present invention that comprisefurin-cleavage resistant, Shiga toxin effector polypeptides exhibitimproved tolerability (e.g. improved non-specific toxicity profiles) ascompared to related, reference molecules comprising wild-type Shigatoxin A1 fragments (see e.g. WO 2015/191764). Such improved in vivotolerability may be due to the increased stability of a linkage betweena Shiga toxin effector polypeptide and a binding region and/or toxiccomponent of the cell-targeting molecule.

C. Testing the Targeted Cytotoxicity and Efficacy of Exemplary,Cell-Targeting Molecules of the Present Invention In Vivo Using AnimalModels

Animal models are used to determine the in vivo effects of exemplary,Shiga toxin effector polypeptides combo(n) and cell-targeting moleculescomprising the aforementioned to target positive, neoplastic cells.Various mice strains are used to test the effects of the cell-targetingmolecules on xenograft tumors in mice after intravenous administrationof each molecule to those mice. In certain experiments, a disseminatedxenograft model for human tumors is used to determine the in vivoefficacy of exemplary, cell-targeting molecules of the present inventionin human-tumor bearing mice. Human tumor cells that constitutivelyexpress luciferase and display cell-surface expression of the target ofthe appropriate scFv-(n) are used in this xenograft model. Methods knownto the skilled worker are used to test the targeted cytotoxicity ofmolecules of the present invention (see e.g. WO 2014/164680, WO2014/164693, WO 2015/191764). Certain cell-targeting molecules of thepresent invention are capable of significantly reducing the human tumorburden in mice challenged with human tumor cells.

As shown in Examples 1-4, combination Shiga toxin A Subunit effectorpolypeptides of the present invention may be used as scaffolds to createcell-targeting molecules that exhibit: 1) increased stability, 2)improved in vivo tolerability in chordates, 2) reduced immunogenicpotential after administration to chordates, and/or the ability todeliver an embedded or inserted T-cell epitope for MHC class Ipresentation by a nucleated, chordate cell. In certain combinations, theresulting level of de-immunization represents the synergistic action ofindividually de-immunized sub-regions which were combined together.

Example 4. An Exemplary Cell-Targeting Molecule Targeting CD38+ Cells

The binding characteristics of the “SLT-1A-combo7::αCD38-scFv-1” protein(SEQ ID NO:82) to an extracellular, human CD38 target was determined bya fluorescence-based, flow-cytometry assay. Samples containing CD38positive (CD38+) cells (of either the cell line A or F) were suspendedin 1×PBS containing one percent bovine serum albumin (BSA) (Calbiochem,San Diego, Calif., U.S.), hereinafter referred to as “1×PBS+1% BSA”, andincubated for one hour at 4° C. with 100 μL of various dilutions ofSLT-1A-combo7:: αCD38-scFv-1. The highest concentrations ofSLT-1A-combo7:: αCD38-scFv-1 tested in the assay was selected tosaturate all of the binding possibilities. CD38 negative (CD38−) cells(of the cell lines H and I) were treated with the highest concentrationof SLT-1A-combo7:: αCD38-scFv-1 tested on target positive cells in thisassay. After the one hour incubation, the cell samples were washed twicewith 1×PBS+1% BSA. The cell samples were incubated for one hour at 4° C.with 100 μL of 1×PBS+1% BSA containing α-SLT-1A pAb1, then washed againand incubated for one hour at 4° C. with 100 μL of 1×PBS+1% BSA solutioncontaining an anti-rabbit secondary antibody conjugated to fluoresceinisothiocyanate (FITC).

The cell samples were washed twice with 1×PBS+1% BSA, resuspended in 200μL of 1×PBS and subjected to fluorescence-based, flow cytometry to assaythe percentage of cells bound by sufficient secondary antibody,indicative of the binding levels of SLT-1A-combo7:: αCD38-scFv-1 to thecells in each sample. The data for all the samples in mean fluorescenceintensity units (MFI), in relative fluorescence units, was obtained bygating the data using a negative control sample of cells which was nottreated with any cell-targeting molecule but which was incubated withthe α-SLT-1A pAb1 primary antibody and the anti-rabbit secondaryantibody as described above. The integrated MFI (iMFI) was calculated bymultiplying the percentage of positive cells with the MFI. Graphs wereplotted of iMFI versus “concentration of protein” in nanomolar usingPrism software (GraphPad Software, San Diego, Calif., U.S.). Using thePrism software function of one-site binding [Y=B_(max)*X/(K_(D)+X)]under the heading binding-saturation, the B_(max) and K_(D) werecalculated using baseline corrected data. B_(max) is the maximumspecific binding reported in iMFI. K_(D) is the equilibrium bindingconstant, reported in nanomolar.

The B_(max) for SLT-1A-combo7:: αCD38-scFv-1 (SEQ ID NO:82) binding totwo, different, CD38+ cell types was measured to be approximately100,000 iMFI, and the K_(D) of SLT-1A-combo7:: αCD38-scFv-1 bindingthose CD38+cells was measured to be approximately 2-7 nM (Table 19; FIG.18). SLT-1A-combo7:: αCD38-scFv-1 did not exhibit specific binding toCD38-cells or high affinity binding to CD38-cells in this assay underthe conditions described (FIG. 18).

TABLE 19 Binding Characteristics: Representative Values for B_(max) andK_(D) for Cell Binding by an Exemplary, Cell- Targeting Molecule of thePresent Invention target positive target positive cell line A cell lineF B_(max) K_(D) Bmax K_(D) Cell-Targeting Molecule (iMFI) (nM) (iMFI)(nM) SLT-1A-combo7::αCD38-scFv-1 112,660 7.1 93,966 1.6

The exemplary, CD38-targeted, cell-targeting molecule SLT-1A-combo7::αCD38-scFv-1 was tested for catalytic activity as described in Example2. SLT-1A-combo7:: αCD38-scFv-1 (SEQ ID NO:82) exhibited ribosomeinactivation activity comparable to a wild-type.

The exemplary, CD38-targeted, cell-targeting molecule SLT-1A-combo7::αCD38-scFv-1 was tested for cytotoxicity as described in Example 2.SLT-1A-combo7:: αCD38-scFv-1 (SEQ ID NO:82) was potently cytotoxic toCD38+cells.

The exemplary, CD38-targeted, cell-targeting molecule SLT-1A-combo7::αCD38-scFv-1 was tested for reduced immunogenicity as described inExample 2. SLT-1A-combo7:: αCD38-scFv-1 (SEQ ID NO:82) exhibited reducedimmunogenicity as compared to a control SLT-1A-FR:: αCD38-scFv-1.

The exemplary, CD38-targeted, cell-targeting molecule SLT-1A-combo7::αCD38-scFv-1 was tested in a xenograft model of human cancer using humancells expressing CD38 and an assay known to the skilled worker (see e.g.WO 2014/164693). SLT-1A-combo7:: αCD38-scFv-1 (SEQ ID NO:82) exhibitedthe ability to reduce tumor burden in mice injected with CD38 positive,human, neoplastic cells as compared to a control, vehicle treated group.In this study, mice were injected with 2.5×10⁶ Daudi-Luc cells (a human,CD38 positive tumor cell line that has been engineered to express theluciferase gene). Four days after tumor injection, mice were randomizedinto groups of eight mice per group and treatment was started. The micein the control and cell-targeting molecule treatment groups receivedvehicle only or cell-targeting molecule, respectively, for twelve dosesover five weeks (three times per week for two weeks, a week of no dosingthen three times a week again for 2 weeks). On different days of thestudy, whole body bioluminescence (BLI) was measured in photons persecond to monitor the tumor burden over time by detecting luciferaseexpressing Daudi cells. Before treatment, the mice were randomized usingtheir BLI readings in order to create mice groups with similar mean andmedian BLI values. The results of this study are reported in Table 20and FIG. 19. Table 20 lists the median BLI signal for the cell-targetingmolecule treated group and the vehicle-only control group and thepercent of “treated over control” (% T/C), defined by the formula:(median BLI signal of the treated group)/(median BLI signal of thevehicle-only control group)×100. Day 4 involved pre-treatmentmeasurements.

TABLE 20 Treatment with the Exemplary, Cell-Targeting Molecule SLT-1A-combo7::αCD38-scFv-1 Reduced Tumor Burdens In Vivo Treatment GroupSLT-1A-combo7::αCD38- cell-targeting scFv-1 molecule vehicle-only BLIPercentage of the cell- Day of median BLI signal targeting moleculetreated Study (photons/second) over control treated (% T/C) 4 1.37e61.46e6 94% 7 1.64e6 2.10e6 78% 14 7.66e6 9.36e7  8% 22 3.60e8 4.22e9  9%28 1.88e9 9.32e9 20% 35  1.24e10  3.26e10 38% 40  1.65e10  1.55e11 11%

BLI measurements taken on study Days 7, 14, 22, 36, and 40 showed thatthe control, vehicle-only group had a higher tumor burden than the grouptreated with 0.5 milligrams of SLT-1A-combo7:: αCD38-scFv-1 per kg ofbody weight per dose. At the final time-point (Day 40), the percentageof BLI for the treated over the vehicle-only control (T/C) was 11%,indicating that there was an 89% reduction of the median tumor burden inthe group treated with SLT-1A-combo7:: αCD38-scFv-1 (SEQ ID NO:82).

Example 5. Cell-Targeting Molecules Comprising a Shiga Toxin EffectorPolypeptide of the Present Invention Linked to a HER2 Binding Region(αHER2-V_(H)H Fused with SLT-1A-Combo(n))

In this example, any one of the Shiga toxin effector polypeptidesSLT-1A-combo0-26, as described above, is operably linked to animmunoglobulin binding region anti-HER2, which binds specifically andwith high-affinity to an extracellular antigen on human HER2, such asthe single-domain variable region of the camelid, antibody 5F7(αHER2-V_(H)H) as described in U.S. Patent Application Publication2011/59,090, to form an exemplary, cell-targeting molecule of thepresent invention.

Construction, Production, and Purification of the Cytotoxic,HER2-Binding, Fusion Protein “SLT-1A-Combo(n)::αHER2-V_(H)H”

For certain embodiments, the immunoglobulin-derived binding regionαHER2-V_(H)H and Shiga toxin effector polypeptide are fused together toform a single, continuous polypeptide “SLT-1A-combo(n)::αHER2-V_(H)H.”In this example, a polynucleotide encoding αHER2-V_(H)H, a bindingregion comprising a V_(H)H variable domain derived from theimmunoglobulin 5F7, is cloned in frame with a polynucleotide encoding alinker known in the art and in frame with a polynucleotide encoding theShiga toxin effector polypeptide SLT-1A-combo(n). Expression of thecell-targeting molecule SLT-1A-combo(n)::αHER2-V_(H)H is accomplishedusing either bacterial and/or cell-free, protein translation systemsknown to the skilled worker and/or as described in the previousexamples.

Determining the In Vitro Characteristics ofSLT-1A-Combo(n)::αHER2-V_(H)H

The binding characteristics of the cell-targeting molecule of thisexample for HER2+cells and HER2− cells is determined by afluorescence-based, flow-cytometry. The B_(max) forSLT-1A-combo(n)::αHER2-V_(H)H to HER2+cells is measured to beapproximately 50,000-200,000 MFI with a K_(D) within the range of0.01-100 nM, whereas there is no significant binding to HER2− cells inthis assay.

The ribosome inactivation abilities of the SLT-1A-combo(n)::αHER2-V_(H)Hcell-targeting molecule is determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cell-targeting molecule of this example on cell-freeprotein synthesis is significant. The IC₅₀ ofSLT-1A-combo(n)::αHER2-V_(H)H on protein synthesis in this cell-freeassay is approximately 0.1-100 μM.

Determining the Cytotoxicity of SLT-1A-Combo(n)::αHER2-V_(H)H Using aHER2+ Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A-combo(n)::αHER2-V_(H)H isdetermined by the general cell-kill assay as described above in theprevious examples using HER2+cells. In addition, the selectivecytotoxicity characteristics of SLT-1A-combo(n)::αHER2-V_(H)H isdetermined by the same general cell-kill assay using HER2− cells as acomparison to the HER2+cells. The CD50 of the cell-targeting molecule ofthis example is approximately 0.01-100 nM for HER2+cells depending onthe cell line. The CD₅₀ of SLT-1A-combo(n)::αHER2-V_(H)H isapproximately 10-10,000 fold greater (less cytotoxic) for cells notexpressing HER2 on a cellular surface as compared to cells which doexpress HER2 on a cellular surface. In addition, the cytotoxicity ofSLT-1A-combo(n)::αHER2-V_(H)H is investigated for both directcytotoxicity and indirect cytotoxicity by T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity using assays known tothe skilled worker and/or described herein.

Determining the In Vivo Effects of the Exemplary, Cell-TargetingMolecule SLT-1A-Combo(n)::αHER2-V_(H)H Using Animal Models

Animal models are used to determine the in vivo effects of thecell-targeting molecule SLT-1A-combo(n)::αHER2-V_(H)H toward neoplasticcells. Various mice strains are used to test the effect ofSLT-1A-combo(n)::αHER2-V_(H)H after intravenous administration onxenograft tumors in mice resulting from the injection into those mice ofhuman neoplastic cells which express HER2 on their cell surfaces. Cellkilling is investigated for both direct cytotoxicity and indirectcytotoxicity by T-cell epitope delivery and presentation leading toCTL-mediated cytotoxicity using assays known to the skilled workerand/or described herein.

Example 6. Cell-Targeting Molecules Comprising a Shiga Toxin a SubunitEffector Polypeptide of the Present Invention, a Fused, T-CellEpitope-Peptide, and a Ligand Binding Region Specific to IL-2R

In this example, the Shiga toxin effector polypeptide is derived fromthe A subunit of Shiga-like Toxin 1 (SLT-1A) as described above,optionally with amino acid residue substitutions conferringfurin-cleavage resistance, such as, e.g., R248A/R251A (see Example 3,supra). A human, CD8+ T-cell epitope-peptide is selected based on MHC Imolecule binding predictions, HLA types, already characterizedimmunogenicities, and readily available reagents as described above,such as the epitope GVMTRGRLK (SEQ ID NO:560). A binding region isderived from a ligand (the cytokine interleukin 2 or IL-2) for the humaninterleukin 2 receptor (IL-2R), which is capable of specifically bindingan extracellular part of the human IL-2R. IL-2R is a cell-surfacereceptor expressed by various immune cell types, such as T-cells andnatural killer cells.

Construction, Production, and Purification of the Cell-Targeting FusionProteins T-Epitope:: SLT-1A::IL-2 and IL-2::T-Epitope::SLT-1A

The ligand-type binding region αIL-2R, the Shiga toxin effectorpolypeptide, and T-cell epitope are linked together to form a single,continuous polypeptide, such as “T-epitope::SLT-1A::IL-2” or“IL-2::T-epitope::SLT-1A,” and, optionally, a KDEL (SEQ ID NO: 514) isadded to the carboxy-terminus of the resulting polypeptide. For example,fusion proteins are produced by expressing from polynucleotides encodingT-epitope::SLT-1A::IL-2 and IL-2::T-epitope::SLT-1A. Expression of theT-epitope::SLT-1A::IL-2 or IL-2::T-epitope::SLT-1A cell-targetingmolecules is accomplished using either bacterial and/or cell-free,protein translation systems as described in the previous examples.

Determining the In Vitro Characteristics of the Cell-Targeting MoleculesSLT-1A::T-Epitope::IL-2 and IL-2::SLT-1A::T-Epitope

The binding characteristics of cell-targeting molecules of this examplefor IL-2R positive cells and IL-2R negative cells is determined byfluorescence-based, flow-cytometry. The Bmax for bothT-epitope::SLT-1A::IL-2 and IL-2::T-epitope::SLT-1A to IL-2R positivecells is measured to be approximately 50,000-200,000 MFI with a K_(D)within the range of 0.01-100 nM, whereas there is no significant bindingto IL-2R negative cells in this assay.

The ribosome inactivation abilities of T-epitope::SLT-1A::IL-2 andIL-2::T-epitope::SLT-1A are determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cell-targeting molecules of this example on cell-freeprotein synthesis is significant. For both T-epitope::SLT-1A::IL-2 andIL-2::T-epitope::SLT-1A, the IC₅₀ of on protein synthesis in thiscell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cell-Targeting Molecules T-Epitope::SLT-1A::IL-2 or IL-2::T-Epitope::SLT-1A Using a Cell-Kill Assay

The cytotoxicity characteristics of T-epitope::SLT-1A::TL-2 orIL-2::T-epitope::SLT-1A are determined by the general cell-kill assay asdescribed above in the previous examples using IL-2R positive cells. Inaddition, the selective cytotoxicity characteristics ofT-epitope::SLT-1A::IL-2 or IL-2::T-epitope::SLT-1A are determined by thesame general cell-kill assay using IL-2R negative cells as a comparisonto the IL-2R positive cells. The CD₅₀ of the cell-targeting molecules ofthis example is approximately 0.01-100 nM for IL-2R positive cellsdepending on the cell line. The CD₅₀ of T-epitope::SLT-1A::IL-2 orIL-2::T-epitope::SLT-1A is approximately 10-10,000 fold greater (lesscytotoxic) for cells not expressing IL-2R on a cellular surface ascompared to cells which do express TL-2R on a cellular surface. Inaddition, the cytotoxicity of T-epitope::SLT-1A::IL-2 orIL-2::T-epitope::SLT-1A is investigated for both direct cytotoxicity andindirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity using assays known to the skilledworker and/or described herein.

Determining the In Vivo Effects of the Cell-Targeting MoleculesT-Epitope::SLT-1A::IL-2 or IL-2::T-Epitope::SLT-1A Using Animal Models

Animal models are used to determine the in vivo effects of thecell-targeting molecules T-epitope::SLT-1A::IL-2 andIL-2::T-epitope::SLT-1A on neoplastic cells. Various mice strains areused to test the effect of T-epitope::SLT-1A::IL-2 andIL-2::T-epitope::SLT-1A after intravenous administration on xenografttumors in mice resulting from the injection into those mice of humanneoplastic cells which express IL-2R on their cell surfaces. Cellkilling is investigated for both direct cytotoxicity and indirectcytotoxicity by T-cell epitope delivery and presentation leading toCTL-mediated cytotoxicity using assays known to the skilled workerand/or described herein.

Example 7. Cell-Targeting Molecules Comprising a Shiga Toxin EffectorPolypeptide of the Present Invention Linked to a CEA Binding Region(αCEA-(Fn3) Binding Region Fused with SLT-1A-Combo(n))

In this example, the Shiga toxin effector polypeptide SLT-1A-combo(n),as described above, is operably linked to an immunoglobulin-type,binding region anti-CEA (Fn3) binding region, which binds specificallyand with high-affinity to an extracellular antigen on humancarcinoembryonic antigen (CEA), such as the tenth human fibronectin typeIII domain derived binding region C743 as described in Pirie C et al., JBiol Chem 286: 4165-72 (2011), to form an exemplary, cell-targetingmolecule of the present invention. In addition, an immunogenic, CD8+T-cell epitope is fused to the amino-terminus of the Shiga toxineffector polypeptide of this example to form epitope-SLT-1A-combo(n).

Construction, Production, and Purification of the Cytotoxic,CEA-Binding, Fusion Protein “αCEA-(Fn3) Fused withEpitope-SLT-1A-Combo(n)”

For certain embodiments, the immunoglobulin-type binding regionαCEA-(Fn3) and Shiga toxin effector polypeptide are fused together toform a single, continuous polypeptide “αCEA-(Fn3) fused withepitope-SLT-1A-combo(n).” In this example, a fusion protein “αCEA-(Fn3)fused with epitope-SLT-1A-combo(n)” is designed and produced asdescribed in the previous examples.

Determining the In Vitro Characteristics of “αCEA-(Fn3) Fused withEpitope-SLT-1A-Combo(n)”

The binding characteristics of the cell-targeting molecule of thisexample for CEA+cells and CEA− cells is determined by afluorescence-based, flow-cytometry. The B_(max) for αCEA-(Fn3) fusedwith epitope-SLT-1A-combo(n) to CEA+cells is measured to beapproximately 50,000-200,000 MFI with a K_(D) within the range of0.01-100 nM, whereas there is no significant binding to CEA− cells inthis assay.

The ribosome inactivation abilities of the αCEA-(Fn3) fused withepitope-SLT-1A-combo(n) cell-targeting molecule is determined in acell-free, in vitro protein translation as described above in theprevious examples. The inhibitory effect of the cell-targeting moleculeof this example on cell-free protein synthesis is significant. The IC₅₀of αCEA-(Fn3) fused with epitope-SLT-1A-combo(n) on protein synthesis inthis cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of “αCEA-(Fn3) Fused withEpitope-SLT-1A-Combo(n)” Using a CEA+ Cell-Kill Assay

The cytotoxicity characteristics of αCEA-(Fn3) fused withepitope-SLT-1A-combo(n) is determined by the general cell-kill assay asdescribed above in the previous examples using CEA+cells. In addition,the selective cytotoxicity characteristics of αCEA-(Fn3) fused withepitope-SLT-1A-combo(n) is determined by the same general cell-killassay using CEA− cells as a comparison to the CEA+cells. The CD₅₀ of thecell-targeting molecule of this example is approximately 0.01-100 nM forCEA+cells depending on the cell line. The CD₅₀ of αCEA-(Fn3) fused withepitope-SLT-1A-combo(n) is approximately 10-10,000 fold greater (lesscytotoxic) for cells not expressing CEA on a cellular surface ascompared to cells which do express CEA on a cellular surface. Inaddition, the cytotoxicity of αCEA-(Fn3) fused withepitope-SLT-1A-combo(n) is investigated for both direct cytotoxicity andindirect cytotoxicity by T-cell epitope delivery and presentationleading to CTL-mediated cytotoxicity using assays known to the skilledworker and/or described herein.

Determining the In Vivo Effects of the Exemplary, Cell-TargetingMolecule “αCEA-(Fn3) Fused with Epitope-SLT-1A-Combo(n)” Using AnimalModels

Animal models are used to determine the in vivo effects of thecell-targeting molecule αCEA-(Fn3) fused with epitope-SLT-1A-combo(n)toward neoplastic cells. Various mice strains are used to test theeffect of αCEA-(Fn3) fused with epitope-SLT-1A-combo(n) afterintravenous administration on xenograft tumors in mice resulting fromthe injection into those mice of human neoplastic cells which expressCEA on their cell surfaces. Cell killing is investigated for both directcytotoxicity and indirect cytotoxicity by T-cell epitope delivery andpresentation leading to CTL-mediated cytotoxicity using assays known tothe skilled worker and/or described herein.

Example 8. Cytotoxic, Cell-Targeting Molecules Comprising a Shiga ToxinEffector Polypeptide SLT-1A-Combo(n) and the AntibodyαHelminth-Intestinal-Antigen

In this example, any on the Shiga toxin effector polypeptidesSLT-1A-combo0-26, as described above, is operationally linked to animmunoglobulin-type binding region targeting a helminth antigen. Animmunoglobulin-type binding region αhelminth-intestinal-antigen isderived from an antibody generated, using techniques known in the art,to the helminth ortholog of a human transferrin receptor (see e.g. thenematode gene gcp-2.1 UniProt G8JYE4_CAEEL; Rosa B et al., Mol CellProteomics M114.046227 (2015)).

Construction, Production, and Purification of the Cytotoxic,Cell-Targeting Molecule “SLT-1A-combo(n):: αHelminth-Intestinal-Antigen”

The immunoglobulin-type binding region αhelminth-intestinal-antigen andShiga toxin effector region, which is optionally a protease-cleavageresistant Shiga toxin effector region, are linked together to form aprotein in which the immunoglobulin-type binding region is not locatedproximally to the protein's amino-terminus as compared to the Shigatoxin effector region. For example, a fusion protein is produced byexpressing a polynucleotide encoding anαhelminth-intestinal-antigen-binding protein fused to an amino-terminalSLT-1A-combo0-26. Expression of theSLT-1A-combo(n)::αhelminth-intestinal-antigen-binding protein isaccomplished using either bacterial and/or cell-free, proteintranslation systems as described in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic,Cell-Targeting Molecule SLT-1A-Combo(n):: αHelminth-Intestinal-Antigen

The binding characteristics of the cytotoxic, cell-targeting molecule ofthis example is determined by a molecular binding assay known in the artusing a purified, recombinant, target protein. The K_(D) forSLT-1A::αhelminth-intestinal-antigen and/orSLT-1A-FR::αhelminth-intestinal-antigen to target protein is measured tobe approximately 100 nM, whereas there is no significant binding to anegative control protein (e.g. purified, recombinant, helminth orthologof human transferrin receptor) in this assay.

The ribosome inactivation abilities of theSLT-1A::αhelminth-intestinal-antigen is determined in a cell-free, invitro protein translation as described above in the previous examples.The inhibitory effect of the cytotoxic, cell-targeting molecule of thisexample on cell-free protein synthesis is significant. The IC₅₀ ofSLT-1A::αhelminth-intestinal-antigen on protein synthesis in thiscell-free assay is approximately 0.1-100 pM.

Determining the Toxicity of the Cytotoxic, Cell-Targeting MoleculeSLT-1A-combo(n)::αHelminth-Intestinal-Antigen

The toxicity of SLT-1A:: αhelminth-intestinal-antigen to helminths isdetermined using model helminths (see e.g. Iatsenko I et al., Toxins2050-63 (2014)). The helminth can be administered purifiedSLT-1A::αhelminth-intestinal-antigen by soaking or alternatively byfeeding the helminth with bacteria expressing the SLT-1A::αhelminth-intestinal-antigen fusion protein.

In addition, laboratory animals harboring helminths and/or displayinghelminth-related diseases are administered SLT-1A::αhelminth-intestinal-antigen and monitored for reduction or eliminationof helminths and/or associated symptoms of parasitic helminth(s).

Example 9. Cell-Targeting Molecules Comprising a Shiga Toxin EffectorPolypeptide SLT-1A-Combo(n) Fused to an Immunoglobulin-Type BindingRegion Specific to HIV-1 Gag

In this example, any one of the Shiga toxin effector polypeptideSLT-1A-combo0-26, as described above, is operably linked to animmunoglobulin-type binding region αGag-antigen, which is derived froman immunoglobulin-type domain recognizing the HIV capsid protein HIV-1Gag polyprotein (see e.g. Nagola S et al., Retrovirology 9: 17 (2012))and which comprises an artificial, Ankyrin domain repeat polypeptidecapable of binding an extracellular part of Gag. Gag is the majorstructural protein involved in HIV virus assembly and oligomerizes intoa lattice of as many as 700 to 5000 copies per virion to form aconical-shaped CA core (see e.g. Chen Y et al., Biophys J 96: 1961-9(2009); Pornillos O et al., Nature 469: 424-7 (2011)).

Construction, Production, and Purification of the Cell-TargetingMolecules “SLT-1A-Combo(n) Linked to αGag”

The immunoglobulin-type binding region αGag and Shiga toxin effectorpolypeptide SLT-1A-combo(n) are fused to form a cytotoxic protein. Forexample, a fusion protein is produced by expressing a polynucleotideencoding the αGag-antigen-binding protein SLT-1A-combo(n)::αGag.Expression of the SLT-1A-combo(n)::αGag cytotoxic protein isaccomplished using either bacterial and/or cell-free, proteintranslation systems as described in the previous examples.

Determining the In Vitro Characteristics of the Cell-Targeting Molecule“SLT-1A-Combo(n)::αGag”

The binding characteristics of the cytotoxic protein of this example forpurified, recombinant HIV-1 Gag is determined by an assay known in theart, such as an enzyme-linked immunosorbent assay. The K_(D) forSLT-1A-combo(n)::αGag binding to Gag is measured to be approximatelywithin the range of 0.01-100 nanomolar (nM).

The ribosome inactivation abilities of the SLT-1A-combo(n)::αGagcytotoxic protein is determined in a cell-free, in vitro proteintranslation as described above in the previous examples. The inhibitoryeffect of the cytotoxic protein of this example on cell-free proteinsynthesis is significant. The IC₅₀ of SLT-1A-combo(n)::αGag on proteinsynthesis in this cell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the Cell-Targeting Molecule“SLT-1A-combo(n)::αGag” Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A-combo(n)::αGag are determinedby the general cell-kill assay as described above in the previousexamples using HIV-infected human T-cells. In addition, the selectivecytotoxicity characteristics of SLT-1A-combo(n)::αGag are determined bythe same general cell-kill assay using uninfected T-cells as acomparison to the infected T-cells. The CD₅₀ of the cytotoxic protein ofthis example is approximately 0.01-100 nM for infected T-cells withactively replicating virus. The CD₅₀ of the cytotoxic protein isapproximately 10-10,000 fold greater (less cytotoxic) for uninfectedT-cells.

Determining the In Vivo Effects of the Cytotoxic, Cell-TargetingMolecule “SLT-1A-Combo(n)::αGag” Using Animal Models

The use of SLT-1A-combo(n)::αGag to inhibit the progression of HIVinfection is tested by administering SLT-1A-combo(n)::αGag to simianimmunodeficiency virus (SIV) infected non-human primates (see Sellier Pet al., PLoS One 5: e10570 (2010)).

Example 10. A Cytotoxic, Cell-Targeting Molecule Derived from the ASubunit of Shiga Toxin and the Antibody αHistoplasma-Antigen

In this example, any one of the Shiga toxin effector polypeptidesSLT-1A-combo0-26, as described above, is operably linked to animmunoglobulin-type binding region αhistoplasma-antigen, which isderived from a known antibody or an antibody generated, using techniquesknown in the art, to a Histoplasma capsulatum surface antigen (see e.g.,H. capsulatum H antigen (Deepe G, Durose G, Infect Immun 63: 3151-7(1995)) and the mAb H1C (Lopes L et al., Clin Vaccine Immunol 17: 1155-8(2010); H. capsulatum, cell surface, histone-like protein H2B (NosanchukJ et al., J Clin Invest 112: 1164-1175 (2003))).

Construction, Production, and Purification of the CytotoxicCell-Targeting Molecule SLT-1A-combo(n):: αHistoplasma-Antigen

The immunoglobulin-type binding region αhelminth-intestinal-antigen andShiga toxin effector polypeptide, which is optionally aprotease-cleavage resistant Shiga toxin effector region, are linkedtogether to form a protein in which the immunoglobulin-type bindingregion is not located proximally to the protein's amino-terminus ascompared to the Shiga toxin effector polypeptide. For example, a fusionprotein is produced by expressing a polynucleotide encoding aHistoplasma-surface-antigen-binding protein fused to an amino-terminal,SLT-1A-combo(n). Expression of theSLT-1A-combo(n)::αHistoplasma-antigen-binding protein is accomplishedusing either bacterial and/or cell-free, protein translation systems asdescribed in the previous examples.

Determining the In Vitro Characteristics of the Cytotoxic,Cell-Targeting Molecule SLT-1A-Combo(n):: αHistoplasma-Antigen

The binding characteristics of the cytotoxic, cell-targeting molecule ofthis example is determined by a molecular binding assay known in the artusing a purified recombinant target protein. The K_(D) forSLT-1A-combo(n)::αHistoplasma-antigen to target protein (e.g. apurified, recombinant, H. capsulatum surface antigen) is measured to beapproximately 100 nM, whereas there is no significant binding to anegative control protein in this assay.

The ribosome inactivation abilities of theSLT-1A-combo(n)::αHistoplasma-antigen cytotoxic protein is determined ina cell-free, in vitro protein translation as described above in theprevious examples. The inhibitory effect of the cytotoxic,cell-targeting molecule of this example on cell-free protein synthesisis significant. The IC₅₀ of SLT-1A-combo(n)::αHistoplasma-antigen onprotein synthesis in this cell-free assay is approximately 0.1-100 pM.

Determining the Anti-Fungal Activity of the Cytotoxic, Cell-TargetingMolecule SLT-1A-Combo(n)::αHistoplasma-Antigen Using Fungi

The fungicidal activity of SLT-1A-combo(n)::αHistoplasma-antigen tofungal cells is determined. Purified, StxA::αHistoplasma-antigen and/orStxA-FR::αHistoplasma-antigen is directly administered to fungalcultures in order to measure fungicidal activity (see e.g. Li R et al.,Antimicrob Agents Chemother 44: 1734-6 (2000)). In addition, laboratoryanimals infected with fungi (e.g. H. capsulatum) and/or displayinghistoplasmosis, systemic mycoses, and/or other H. capsulatum-relateddiseases are administered SLT-1A-combo(n)::αHistoplasma-antigen andmonitored for reduction or elimination of fungal pathogens and/orassociated symptoms of fungal infections (see e.g. Kobayashi G et al.,Antimicrob Agents Chemother 34: 524-8 (1990)).

Example 11. Cytotoxic Cell-Targeting Molecules Targeting Various CellTypes

In this example, the Shiga toxin effector region is derived from the Asubunit of Shiga-like Toxin 1 (SLT-1A), Shiga toxin (StxA), and/orShiga-like Toxin 2 (SLT-2A) such that it comprises a combination ofsub-regions described herein to provide two or more of the following: 1)de-immunization, 2) protease-cleavage resistance, and/or 3) an embeddedor inserted, heterologous, T-cell epitope. A binding region is derivedfrom the molecules chosen from column 1 of Table 21 and which binds theextracellular target biomolecule indicated in column 2 of Table 21. Theresulting combination, Shiga toxin effector polypeptides and bindingregions are fused together to from various, single-chain polypeptides.The exemplary proteins of this example are optionally created with acarboxy-terminal KDEL-type signal motif using techniques known in theart and optionally linked to an additional exogenous material, such as,a detection promoting agent(s). The exemplary proteins of this exampleare tested as described in the previous examples using cells expressingthe appropriate extracellular target biomolecules. The exemplaryproteins of this example may be used, e.g., to labeling subcellularcompartments of target cells and to diagnose and treat diseases,conditions, and/or disorders indicated in column 3 of Table 21.

TABLE 21 Various Binding Regions for Cell-Targeting of CytotoxicProteins Source of Extracellular binding region target Application(s)alemtuzumab CD52 B-cell cancers, such as lymphoma and leukemia, andB-cell related immune disorders, such as autoimmune disordersbasiliximab CD25 T-cell disorders, such as prevention of organtransplant rejections, and some B-cell lineage cancers brentuximab CD30hematological cancers, B-cell related immune disorders, and T-cellrelated immune disorders catumaxomab EpCAM various cancers, such asovarian cancer, malignant ascites, gastric cancer cetuximab EGFR variouscancers, such as colorectal cancer and head and neck cancer daclizumabCD25 B-cell lineage cancers and T-cell disorders, such as rejection oforgan transplants daratumumab CD38 hematological cancers, B-cell relatedimmune disorders, and T-cell related immune disorders dinutuximabganglioside Various cancers, such as breast cancer, myeloid GD2 cancers,and neuroblastoma efalizumab LFA-1 autoimmune disorders, such aspsoriasis (CD11a) ertumaxomab HER2/neu various cancers and tumors, suchas breast cancer and colorectal cancer gemtuzumab CD33 myeloid cancer orimmune disorder ibritumomab CD20 B-cell cancers, such as lymphoma andleukemia, and B-cell related immune disorders, such as autoimmunedisorders ipilimumab CD152 T-cell related disorders and various cancers,such as leukemia, melanoma muromonab CD3 prevention of organ transplantrejections natalizumab integrin α4 autoimmune disorders, such asmultiple sclerosis and Crohn's disease obinutuzumab CD20 B-cell cancers,such as lymphoma and leukemia, and B-cell related immune disorders, suchas autoimmune disorders ocaratuzumab CD20 B-cell cancers, such aslymphoma and leukemia, and B-cell related immune disorders, such asautoimmune disorders ocrelizumab CD20 B-cell cancers, such as lymphomaand leukemia, and B-cell related immune disorders, such as autoimmunedisorders ofatumumab CD20 B-cell cancers, such as lymphoma and leukemia,and B-cell related immune disorders, such as autoimmune disorderspalivizumab F protein of treat respiratory syncytial virus respiratorysyncytial virus panitumumab EGFR various cancers, such as colorectalcancer and head and neck cancer pertuzumab HER2/neu various cancers andtumors, such as breast cancer and colorectal cancer pro 140 CCR5 HIVinfection and T-cell disorders ramucirumab VEGFR2 various cancers andcancer related disorders, such as solid tumors rituximab CD20 B-cellcancers, such as lymphoma and leukemia, and B-cell related immunedisorders, such as autoimmune disorders tocilizumab or IL-6 receptorautoimmune disorders, such as rheumatoid atlizumab arthritis tositumomabCD20 B-cell cancers, such as lymphoma and leukemia, and B-cell relatedimmune disorders, such as autoimmune disorders trastuzumab HER2/neuvarious cancers and tumors, such as breast cancer and colorectal cancerublituximab CD20 B-cell cancers, such as lymphoma and leukemia, andB-cell related immune disorders, such as autoimmune disordersvedolizumab integrin α4β7 autoimmune disorders, such as Crohn's diseaseand ulcerative colitis CD20 binding CD20 B-cell cancers, such aslymphoma and leukemia, antibodies and and B-cell related immunedisorders, such as scFv(s) autoimmune disorders (see e.g. Geng S et al.,Cell Mol Immunol 3: 439-43 (2006); Olafesn T et al., Protein Eng Des Sel23: 243-9 (2010)) CD22 binding CD22 B-cell cancers or B-cell relatedimmune disorders scFv(s) (see e.g. Kawas S et al., MAbs 3: 479-86(2011)) CD25 binding CD25 various cancers of the B-cell lineage andimmune scFv(s) disorders related to T-cells (see e.g. Muramatsu H etal., Cancer Lett 225: 225-36 (2005)) CD30 binding CD30 B-cell cancers orB-cell/T-cell related immune monoclonal disorders (see e.g. Klimka A etal., Br J Cancer antibody(ies) 83: 252-60 (2000)) CD33 binding CD33myeloid cancer or immune disorder (see e.g. monoclonal Benedict C etal., J Immunol Methods 201: 223- antibody(ies) 31 (1997)) CD38 bindingCD38 hematological cancers, B-cell related immune immunoglobuldisorders, and T-cell related immune disorders in domains (see e.g. U.S.Pat. No. 8,153,765) CD40 binding CD40 various cancers and immunedisorders (see e.g. scFv(s) Ellmark P et al., Immunology 106: 456-63(2002)) CD45 binding CD45 Hematological cancers and myelodysplasticmonoclonal syndromes (see e.g. Matthews D et al., Blood 94:antibody(ies) 1237-47 (1999); Lin Y et al., Cancer Res 66: and scFv(s)3884-92 (2006); Pagel J et al., Blood 107: 2184- 91 (2006)) CD52 bindingCD52 B-cell cancers, such as lymphoma and leukemia, monoclonal andB-cell related immune disorders, such as antibody(ies) autoimmunedisorders (see e.g. U.S. Pat. No. 7,910,104) CD56 binding CD56 immunedisorders and various cancers, such as monoclonal lung cancer, Merkelcell carcinoma, myeloma antibody(ies) (see e.g. Shin J et al., Hybridoma18: 521-7 (1999)) CD79 binding CD79 B-cell cancers or B-cell relatedimmune disorders monoclonal (see e.g. Zhang L et al., Ther Immunol 2:191- antibody(ies) 202 (1995)) CD133 CD133 various cancers, hematologicmalignancies, and binding immune disorders (see e.g. Bidlingmaier S etal., monoclonal J Mol Med 86: 1025-32 (2008); Pavlon L et al., Jantibodies and Microsc 231: 374-83 (2008); Rappa G et al., scFv(s) StemCells 26: 3008-17 (2008); Swaminathan S et al., J Immunol Methods 361:110-5 (2010); Wang J et al., Hybridoma 29: 241-9 (2010); Zhu X et al.,Mol Cancer Ther 9: 2131-41 (2010); Xia J et al., Sci Rep 3: 3320 (2013))CD248 CD248 various cancers, such as inhibiting angiogenesis binding(see e.g. Zhao A et al., J Immunol Methods 363: scFv(s) 221-32 (2011))EpCAM EpCAM various cancers, such as ovarian cancer, binding malignantascites, gastric cancer (see e.g. monoclonal Schanzer J et al., JImmunother 29: 477-88 antibody(ies) (2006)) PSMA binding PSMA prostatecancer (see e.g. Frigerio B et al., Eur J monoclonal Cancer 49: 2223-32(2013)) antibody(ies) Eph-B2 Eph-B2 various cancers such as colorectalcancer and binding prostate cancer (see e.g. Abéngozar M et al.,monoclonal Blood 119: 4565-76 (2012)) antibody(ies) Endoglin Endoglinvarious cancers, such as breast cancer and binding colorectal cancers(see e.g. Völkel T et al., monoclonal Biochim Biophys Res Acta 1663:158-66 (2004)) antibody(ies) FAP binding FAP various cancers, such assarcomas and bone monoclonal cancers (see e.g. Zhang J et al., FASEB J27: antibody(ies) 581-9 (2013)) CEA binding CEA various cancers, such asgastrointestinal cancer, antibody(ies) pancreatic cancer, lung cancer,and breast cancer and scFv(s) (see e.g. Neumaier M et al., Cancer Res50: 2128-34 (1990); Pavoni E et al., BMC Cancer 6: 4 (2006); Yazaki P etal., Nucl Med Biol 35: 151- 8 (2008); Zhao J et al., Oncol Res 17:217-22 (2008)) CD24 binding CD24 various cancers, such as bladder cancer(see e.g. monoclonal Kristiansen G et al., Lab Invest 90: 1102-16antibody(ies) (2010)) LewisY LewisY various cancers, such as cervicalcancer and antigen antigens uterine cancer (see e.g. Power B et al.,Protein binding Sci 12: 734-47 (2003); Feridani A et al., scFv(s)Cytometry 71: 361-70 (2007)) adalimumab TNF-α various cancers and immunedisorders, such as rheumatoid arthritis, Crohn's Disease, plaquepsoriasis, psoriatic arthritis, ankylosing spondylitis, juvenileidiopathic arthritis, hemolytic disease of the newborn afelimomab TNF-αvarious cancers and immune disorders ald518 IL-6 various cancers andimmune disorders, such as rheumatoid arthritis anrukinzumab IL-13various cancers and immune disorders or ima-638 briakinumab IL-12, IL-23various cancers and immune disorders, such as psoriasis, rheumatoidarthritis, inflammatory bowel diseases, multiple sclerosis brodalumabIL-17 various cancers and immune disorders, such as inflammatorydiseases canakinumab IL-1 various cancers and immune disorders, such asrheumatoid arthritis certolizumab TNF-α various cancers and immunedisorders, such as Crohn's disease fezakinumab IL-22 various cancers andimmune disorders, such as rheumatoid arthritis, psoriasis ganitumabIGF-I various cancers golimumab TNF-α various cancers and immunedisorders, such as rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis infliximab TNF-α various cancers and immune disorders, suchas rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis,psoriasis, Crohn's disease, ulcerative colitis ixekizumab IL-17A variouscancers and immune disorders, such as autoimmune diseases mepolizumabIL-5 various immune disorders and cancers, such as B-cell cancersnerelimomab TNF-α various cancers and immune disorders olokizumab IL6various cancers and immune disorders ozoralizumab TNF-α inflammationperakizumab IL17A various cancers and immune disorders, such asarthritis placulumab human TNF various immune disorders and cancerssarilumab IL6 various cancers and immune disorders, such as rheumatoidarthritis, ankylosing spondylitis siltuximab IL-6 various cancers andimmune disorders sirukumab IL-6 various cancers and immune disorders,such as rheumatoid arthritis tabalumab BAFF B-cell cancers ticilimumabor CTLA-4 various cancers tremelimumab tildrakizumab IL23immunologically mediated inflammatory disorders tnx-650 IL-13 variouscancers and immune disorders, such as B-cell cancers tocilizumab or IL-6receptor various cancers and immune disorders, such as atlizumabrheumatoid arthritis ustekinumab IL-12, IL-23 various cancers and immunedisorders, such as multiple sclerosis, psoriasis, psoriatic arthritisVarious VEGFR, various cancer, such as breast cancer and colon growthEGFR, FGFR cancer, and to inhibit vascularization factors: VEGF, EGF1,EGF2, FGF Various IL-2R, IL-6R, various immune disorders and cancerscytokines: IL- IL-23R, 2, IL-6, IL-23, CD80/CD86, CCL2, TNFRSF13/ BAFFs,TNFs, TNFRSF17, RANKL TNFR Broadly Influenza viral infections (see e.g.Prabakaran P et al., neutralizing surface Front Microbiol 3: 277 (2012))antibodies antigens (e.g. identified from hemaglutinins patient andmatrix samples protein 2) Broadly Coronavirus viral infections (see e.g.Prabakaran P et al., neutralizing surface Front Microbiol 3: 277 (2012))antibodies antigens identified from patient samples Various Filovirusviral infections (see e.g. Olinger G et al., Proc antibodies surfaceNatl Acad Sci USA 109: 18030-5 (2012); Pettitt J antigens (e.g. et al.,Sci Transl Med 5: 199ra113 (2013); VP35, VP40, Stahelin R, Expert OpinTher Targets 18: 115-20 and (2014); Becquart P et al., PLoS One 9:e96360 glycoprotein) (2014); Stahelin R, Fron Microbiol 5: 300 (2014);Tran E et al., J Virol 88: 10958-62 (2014); Murin C et al., Proc NatlAcad Sci USA 111: 17182-7 (2014)) Broadly Henipavirus viral infections(see e.g. Prabakaran P et al., neutralizing surface Front Microbiol 3:277 (2012)) antibodies antigens identified from patient samples VariousHIV surface viral infections (see e.g. Kitidee K et al., BMC antibodiesantigens (e.g. Biotechnol 10: 80 (2010); Yu L, Guan Y, Front includingmatrix protein Immunol 5: 250 (2014)) broadly Map17) neutralizingantibodies and scFvs

While some embodiments of the invention have been described by way ofillustration, it will be apparent that the invention may be put intopractice with many modifications, variations and adaptations, and withthe use of numerous equivalents or alternative solutions that are withinthe scope of persons skilled in the art, without departing from thespirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. The international patent application publications WO2014/164680, WO 2014/164693, WO 2015/138435, WO 2015/138452, WO2015/113005, WO 2015/113007, and WO 2015/191764, are each incorporatedherein by reference in its entirety. The disclosures of U.S. patentapplications US20150259428; 62/168,758; 62/168,759; 62/168,760;62/168,761; 62/168,762; and 62/168,763 are each incorporated herein byreference in its entirety. The disclosure of international PCT patentapplication serial number PCT/US2016/016580 is incorporated herein byreference in its entirety. The complete disclosures of allelectronically available biological sequence information from GenBank(National Center for Biotechnology Information, U.S.) for amino acid andnucleotide sequences cited herein are each incorporated herein byreference in their entirety.

Sequence Listing ID Number Text Description Biological SequenceSEQ ID NO: 1 Shiga-like toxin 1 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSubunit A (SLT- SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIV 1A)ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA VTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISS SEQ ID NO: 2 Shiga toxinKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT Subunit ASLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIV ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILW DSSTLGAILMRRTISS SEQ ID NO: 3Shiga-like toxin 2 DEFTVDFSSQKSYVDSLNSIRSAISTPLGNISQGGV Subunit A (SLT-SVSVINHVLGGNYISLNVRGLDPYSERFNHLRLIM 2A)ERNNLYVAGFINTETNIFYRFSDFSHISVPDVITVS MTTDSSYSSLQRIADLERTGMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFVTVIAEALRFRQIQR GFRPALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEEGVRIGRISFNSLSAILGSVAVILNCHSTGS YSVRSVSQKQKTECQIVGDRAAIKVNNVLWEANTIAALLNRKPQDLTEPNQ SEQ ID NO: 4 Shiga toxinKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT effectorSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIV polypeptide SLT-ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA 1A-WTVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO:5 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTeffector SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIV polypeptide SLT-ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA 1A-FRVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 6 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTeffector SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIV polypeptide SLT-ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTNL 1A-combo0VPMVATVSYTTLQRVAGISRTGMQINRHSLTTSY LDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 7 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTeffector SLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo1TLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 8 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTeffector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo2TLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 9 Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTeffector SLLMIDSGSGDNLFAVNLVPMVATVGRFNNLRLI polypeptide SLT-VERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTT 1A-combo3AVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSY LDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 10effector SLLMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIV polypeptide SLT-ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA 1A-combo4VTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 11effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo5TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 12effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo6TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 13effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo7TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 14effector SLLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo8TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 15effector SLLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo9TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 16effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo10TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 17effector SLLMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo11TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 18effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo12TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 19effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo13TLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGILGDVFTRSYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 20effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo14TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 21effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo15TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 22effector LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER polypeptide SLT-NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVT 1A-combo16LSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDL MSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin AEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 23effector LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER polypeptide SLT-NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVT 1A-combo17LSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDL MSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin AEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 24effector LLMIDSGIGDNLFAVNLVPMVATVGRFNNLRLIV polypeptide SLT-ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA 1A-combo18VTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS NO: 25effector LLMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVER polypeptide SLT-NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVT 1A-combo19LSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDL MSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLAALSGASYVMTAEDVDLTLNWGRLSSV LPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 26effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo20TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHARNLVPMVATVASAVAA SEQ ID Shiga toxinKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 27 effectorSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNATNNVFYRFADFSHVTFPGTTAV 1A-combo21TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 28effector SLLMIDSGSGDNLFAVDVRGIAPEEGRFNNLRLIV polypeptide SLT-ERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTA 1A-combo22VTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYL DLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 29effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAV 1A-combo23TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 30effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo24TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 31effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo25TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLA LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID Shiga toxin KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT NO: 32effector SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVE polypeptide SLT-RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV 1A-combo26TLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLD LMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVALTLNWGRLSS VLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 33 scFv-3::SLT-1A- MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNWT VHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYY GSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQIN RHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNCHHHASRVAR SEQ ID NO: 34SLT-1A-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 1SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 35 SLT-1A-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 2SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 36SLT-1A-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 4SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSA SEQ ID NO: 37 SLT-1A-FR::scFv-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 5SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO: 38 SLT-1A-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 6SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLS CAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 39 SLT-1A-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 7SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPG ASVKMSCKASGYTFTSYVNIHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELS SLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 40 SLT-1A-FR::scFv- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 8SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISCRASQDISNYLAWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA SEQ ID NO: 41 LT-1A-FR::scFv-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 9SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLE WLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTV TVSS SEQ ID NO: 42 SLT-1A-WT::scFv-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT 9SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAREFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGKGLE WLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTV TVSS SEQ ID NO: 43 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo1::scFv-1SLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 44 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo7::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 45 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo8::scFv-1 SLLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 46 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo9::scFv-1 SLLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 47 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo10::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 48 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo11::scFv-1 SLLMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO:49 SLT-1A- MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo12::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 50 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo14::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 51 SLT-1A- MAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo15::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 52 SLT-1A- MKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTScombo16::scFv-1 LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 53 SLT-1A- MAEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTScombo17::scFv-1 LLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 54 SLT-1A- MAEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTScombo18::scFv-1 LLMIDSGIGDNLFAVNLVPMVATVGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 55 SLT-1A- MKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTScombo19::scFv-1 LLMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LAALSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 56 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo20::scFv-1 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHARNLVPMVATVASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQES GPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQ VSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO: 57 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo0::scFv-2 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTNLVPM VATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 58 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo2::scFv-2SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID NO: 59 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo3::scFv-2SLLMIDSGSGDNLFAVNLVPMVATVGRFNNLRLIVE RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFR TTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQP PGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDS WGQGSLVTVSSA SEQ ID NO: 60 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo4::scFv-2SLLMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVE RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFR TTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQP PGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDS WGQGSLVTVSSA SEQ ID NO: 61 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo10::scFv-2SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 62 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo13::scFv-2SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRG ILGDVFTRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSS SEQ ID NO: 63 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo22::scFv-2SLLMIDSGSGDNLFAVDVRGIAPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 64scFv-3::SLT-1A- MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYN combo5VHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGK ATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEG SQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINR HSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNCHHHASRVAR SEQ ID NO: 65scFv-3::SLT-1A- MQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYN combo6VHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGK ATLTADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEG SQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINR HSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI LNCHHHASRVAR SEQ ID NO: 66 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo7::scFv-4SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 67 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo14::scFv-4SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 68 SLT-1A-MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGT combo7::scFv-5SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO: 69 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo8::scFv-5 SLLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO: 70 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo9::scFv-5 SLLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO: 71 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo11::scFv-5 SLLMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRS EDTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO: 72 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo7::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 73 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo8::scFv-6 SLLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 74 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo9::scFv-6 SLLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 75 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo21::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 76 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo23::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 77 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo24::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 78 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo25::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 79 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo26::scFv-6 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGG STYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 80 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo0::scFv-8 SLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTNLVPM VATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISCRASQDISNYLAWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA SEQ ID NO: 81 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo7::scFv-7 SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASS VDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVK PGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYM ELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 82 SLT-1A- MKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTcombo7::αCD38- SLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVER scFv-1NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 83 heavy chain ABR1 YTFTSYVMH SEQ ID NO: 84 heavy chain ABR2WIGYINPYNDGTKY SEQ ID NO: 85 heavy chain ABR3 RGTYYYGSRVFDYSEQ ID NO: 86 light chain ABR1 KSLLNSNGNTYLY SEQ ID NO: 87light chain ABR2 LLIYRMSNLAS SEQ ID NO: 88 light chain ABR3 MQHLEYPFSEQ ID NO: 89 heavy chain ABR1 YAFSSYWMN SEQ ID NO: 90 heavy chain ABR2WIGQIWPGDGDTNY SEQ ID NO: 91 heavy chain ABR3 RRETTTVGRYYYAMDYSEQ ID NO: 92 light chain ABR1 QSVDYDGDSYLN SEQ ID NO: 93light chain ABR2 LLIYDASNLVS SEQ ID NO: 94 light chain ABR3 QQSTEDPWSEQ ID NO: 95 heavy chain ABR2 WIGQIWPGDGDTNYNG SEQ ID NO: 96heavy chain ABR1 GSISTSGMGVG SEQ ID NO: 97 heavy chain ABR2WIGHIWWDDDKRY SEQ ID NO: 98 heavy chain ABR3 RMELWSYYFDY SEQ ID NO: 99light chain ABR1 SSVSYMH SEQ ID NO: 100 light chain ABR2 LLIYDTSKLASSEQ ID NO: 101 light chain ABR3 FQGSVYPF SEQ ID NO: 102 heavy chain CDR1GYTFTSYNMH SEQ ID NO: 103 heavy chain CDR2 AIYPGNGDTSYNQKFKGSEQ ID NO: 104 heavy chain CDR3 AQLRPNYWYFDV SEQ ID NO: 105light chain CDR1 RASSSVSYMH SEQ ID NO: 106 light chain CDR2 ATSNLASSEQ ID NO: 107 light chain CDR3 QQWISNPPT SEQ ID NO: 108heavy chain CDR1 GYTFTSYNVH SEQ ID NO: 109 heavy chain CDR3SNYYGSSYVWFFDV SEQ ID NO: 110 light chain CDR1 RASSSVSYMD SEQ ID NO: 111heavy chain CDR3 STYYGGDWYFNV SEQ ID NO: 112 light chain CDR1 RASSSVSYIHSEQ ID NO: 113 light chain CDR3 QQWTSNPPT SEQ ID NO: 114heavy chain CDR1 GFTFNDYAMH SEQ ID NO: 115 heavy chain CDR2TISWNSGSIGYADSVKG SEQ ID NO: 116 heavy chain CDR3 DIQYGNYYYGMDVSEQ ID NO: 117 light chain CDR1 RASQSVSSYLA SEQ ID NO: 118light chain CDR2 DASNRAT SEQ ID NO: 119 light chain CDR3 QQRSNWPITSEQ ID NO: 120 heavy chain CDR1 GYTFTSYNMH SEQ ID NO: 121heavy chain CDR3 VVYYSNSYWYFDV SEQ ID NO: 122 light chain CDR2 APSNLASSEQ ID NO: 123 light chain CDR3 QQWSFNPPT SEQ ID NO: 124heavy chain CDR1 GYAFSYSWIN SEQ ID NO: 125 heavy chain CDR2RIFPGDGDTDYNGKFKG SEQ ID NO: 126 heavy chain CDR3 NVFDGYWLVYSEQ ID NO: 127 light chain CDR1 RSSKSLLHSNGITYLY SEQ ID NO: 128light chain CDR2 QMSNLVS SEQ ID NO: 129 light chain CDR3 AQNLELPYTSEQ ID NO: 130 heavy chain ABR1 YRFTNYWIH SEQ ID NO: 131heavy chain ABR2 WIGGINPGNNYATYRR SEQ ID NO: 132 heavy chain ABR3TREGYGNYGAWFAY SEQ ID NO: 133 light chain ABR1 QSLANSYGNTFLSSEQ ID NO: 134 light chain ABR2 LLIYGISNRFS SEQ ID NO: 135light chain ABR3 LQGTHQPY SEQ ID NO: 136 heavy chain ABR1 FAFSIYDMSSEQ ID NO: 137 heavy chain ABR2 WVAYISSGGGTTYY SEQ ID NO: 138heavy chain ABR3 RHSGYGTHWGVLFAY SEQ ID NO: 139 light chain ABR1QDISNYLA SEQ ID NO: 140 light chain ABR2 LLIYYTSILHS SEQ ID NO: 141light chain ABR3 QQGNTLPW SEQ ID NO: 142 heavy chain ABR1 YTFTSYWLHSEQ ID NO: 143 heavy chain ABR2 WIGYINPRNDYTEY SEQ ID NO: 144heavy chain ABR3 RRDITTFY SEQ ID NO: 145 light chain ABR1 QSVLYSANHKNYLASEQ ID NO: 146 light chain ABR2 LLIYWASTRES SEQ ID NO: 147light chain ABR3 HQYLSSW SEQ ID NO: 148 heavy chain ABR1 YEFSRSWMNSEQ ID NO: 149 heavy chain ABR2 WVGRIYPGDGDTNYSGKF SEQ ID NO: 150heavy chain ABR3 RDGSSWDWYFDV SEQ ID NO: 151 light chain ABR1QSIVHSVGNTFLE SEQ ID NO: 152 light chain ABR2 LLIYKVSNRFS SEQ ID NO: 153light chain ABR3 FQGSQFPY SEQ ID NO: 154 heavy chain CDR1 GYRFTNYWIHSEQ ID NO: 155 heavy chain CDR2 GINPGNNYATYRRKFQG SEQ ID NO: 156heavy chain CDR3 EGYGNYGAWFAY SEQ ID NO: 157 light chain CDR1RSSQSLANSYGNTFLS SEQ ID NO: 158 light chain CDR2 GISNRFS SEQ ID NO: 159light chain CDR3 LQGTHQPYT SEQ ID NO: 160 heavy chain CDR1 GFAFSIYDMSSEQ ID NO: 161 heavy chain CDR2 YISSGGGTTYYDTVGK SEQ ID NO: 162heavy chain CDR3 HSGYGTHWGVLFAY SEQ ID NO: 163 light chain CDR1RASQDISNYLA SEQ ID NO: 164 light chain CDR2 YTSILHS SEQ ID NO: 165light chain CDR2 QQGNTLPWT SEQ ID NO: 166 heavy chain CDR1 GYTFTDYYITSEQ ID NO: 167 heavy chain CDR2 WIYPGSGNTKYNEKF SEQ ID NO: 168heavy chain CDR3 YGNYWFAY SEQ ID NO: 169 light chain CDR1KASQSVDFDGDSYMN SEQ ID NO: 170 light chain CDR2 AASNLES SEQ ID NO: 171light chain CDR3 QQSNEDPWT SEQ ID NO: 172 heavy chain CDR1 YTFTTYWMHSEQ ID NO: 173 heavy chain CDR2 WIGYINPSTGYTDY SEQ ID NO: 174heavy chain CDR3 TRRGPSYGNHGAWFPY SEQ ID NO: 175 light chain CDR1ENVDTYVS SEQ ID NO: 176 light chain CDR2 LLIYGASNRYT SEQ ID NO: 177light chain CDR3 GQSYRYPP SEQ ID NO: 178 heavy chain CDR1 GYTFTGYYMHSEQ ID NO: 179 heavy chain CDR2 WIDPNSGATTYAQKF SEQ ID NO: 180heavy chain CDR3 KTTQTTWGFPF SEQ ID NO: 181 light chain CDR1 RASQGVYQWLASEQ ID NO: 182 light chain CDR2 KASHLYN SEQ ID NO: 183 light chain CDR3QQLNSYPLT SEQ ID NO: 184 heavy chain CDR1 GYTFTDYWMH SEQ ID NO: 185heavy chain CDR2 WIGYINPNTAYTDY SEQ ID NO: 186 light chain CDR1KASENVDSFVS SEQ ID NO: 187 light chain CDR2 GASNRYT SEQ ID NO: 188light chain CDR3 GQNYRYPLT SEQ ID NO: 189 heavy chain ABR1 FSLISYGVHSEQ ID NO: 190 heavy chain ABR2 WLGVIWRGGSTDY SEQ ID NO: 191heavy chain ABR3 KTLITTGYAMDY SEQ ID NO: 192 light chain ABR1 EDIYNRLASEQ ID NO: 193 light chain ABR2 LLISGATSLETG SEQ ID NO: 194light chain ABR3 QQYWSTP SEQ ID NO: 195 heavy chain ABR1 FTFNSFAMSSEQ ID NO: 196 heavy chain ABR2 WVSAISGSGGGTYY SEQ ID NO: 197heavy chain ABR3 KDKILWFGEPVFDY SEQ ID NO: 198 light chain ABR1 QSVSSYLASEQ ID NO: 199 light chain ABR2 LLIYDASNRAT SEQ ID NO: 200light chain ABR3 QQRSNWPP SEQ ID NO: 201 heavy chain ABR1 FSLTSYGVHSEQ ID NO: 202 heavy chain ABR2 WIGVMWRGGSTDY SEQ ID NO: 203heavy chain ABR3 KSMITTGFVMDS SEQ ID NO: 204 light chain ABR1 EDIYNRLTSEQ ID NO: 205 light chain ABR2 LLISGATSLET SEQ ID NO: 206light chain ABR3 QQYWSNPY SEQ ID NO: 207 heavy chain ABR1 FDFSRSWMNSEQ ID NO: 208 heavy chain ABR2 WIGEINPDSSTINY SEQ ID NO: 209heavy chain ABR3 RYGNWFPY SEQ ID NO: 210 light chain ABR1 QNVDTNVASEQ ID NO: 211 light chain ABR2 ALIYSASYRYS SEQ ID NO: 212light chain ABR3 QQYDSYPL SEQ ID NO: 213 heavy chain ABR1 GTFSSYAFSSEQ ID NO: 214 heavy chain ABR2 WMGRVIPFLGIANS SEQ ID NO: 215heavy chain ABR3 RDDIAALGPFDY SEQ ID NO: 216 light chain ABR1 QGISSWLASEQ ID NO: 217 light chain ABR2 SLIYAASSLQS SEQ ID NO: 218light chain ABR3 QQYNSYPR SEQ ID NO: 219 heavy chain ABR1 YTFTDYWMQSEQ ID NO: 220 heavy chain ABR2 WIGTIYPGDGDTGY SEQ ID NO: 221heavy chain ABR3 RGDYYGSNSLDY SEQ ID NO: 222 light chain ABR1 QDVSTVVASEQ ID NO: 223 light chain ABR2 RLIYSASYRYI SEQ ID NO: 224light chain ABR3 QQHYSPPYYY SEQ ID NO: 225 heavy chain CDR1 GFSLTSYGVHSEQ ID NO: 226 heavy chain CDR2 VMWRGGSTDYNAAFMS SEQ ID NO: 227heavy chain CDR3 SMITTGFVMDS SEQ ID NO: 228 light chain CDR1 KASEDIYNRLTSEQ ID NO: 229 light chain CDR2 GATSLET SEQ ID NO: 230 light chain CDR3QQYWSNPYT SEQ ID NO: 231 heavy chain CDR1 GFSLISYGVH SEQ ID NO: 232heavy chain CDR2 VIWRGGSTDYNAAFMS SEQ ID NO: 233 heavy chain CDR3TLITTGYAMDY SEQ ID NO: 234 light chain CDR1 KASEDIYNRLA SEQ ID NO: 235light chain CDR2 GATSLET SEQ ID NO: 236 light chain CDR3 QQYWSTPTSEQ ID NO: 237 heavy chain CDR1 GFDFSRSWMN SEQ ID NO: 238heavy chain CDR2 EINPDSSTINYTTSLKD SEQ ID NO: 239 heavy chain CDR3YGNWFPY SEQ ID NO: 240 light chain CDR1 KASQNVDTNVA SEQ ID NO: 241light chain CDR2 SASYRYS SEQ ID NO: 242 light chain CDR3 QQYDSYPLTSEQ ID NO: 243 heavy chain ABR1 FDFSRYWMS SEQ ID NO: 244heavy chain ABR2 WIGEINPTSSTINF SEQ ID NO: 245 heavy chain ABR3RGNYYRYGDAMDY SEQ ID NO: 246 light chain ABR1 KSVSTSGYSYLHSEQ ID NO: 247 light chain ABR2 LLIYLASNLES SEQ ID NO: 248light chain ABR3 QHSRELPF SEQ ID NO: 249 heavy chain ABR1 STFTTYWIHSEQ ID NO: 250 heavy chain ABR2 WIGYINPNTGYTEY SEQ ID NO: 251heavy chain ABR3 VRFITVVGG SEQ ID NO: 252 light chain ABR1 SSVSSSHLHSEQ ID NO: 253 light chain ABR2 LWIYSTSNLAS SEQ ID NO: 254light chain ABR3 HQYHRSPL SEQ ID NO: 255 heavy chain ABR1 FSLTTYGIGVGSEQ ID NO: 256 heavy chain ABR2 WLTHIWWNDNKYY SEQ ID NO: 257heavy chain ABR3 YGYTY SEQ ID NO: 258 light chain ABR1 QSLLYSNGNTYLHSEQ ID NO: 259 light chain ABR2 LLIYKLSNRFS SEQ ID NO: 260light chain ABR3 SQSTHVPW SEQ ID NO: 261 heavy chain ABR1 FNIKDTYIHSEQ ID NO: 262 heavy chain ABR2 WVARIYPTNGYTRY SEQ ID NO: 263heavy chain ABR3 RWGGDGFYAMDY SEQ ID NO: 264 light chain ABR1 QDVNTAVASEQ ID NO: 265 light chain ABR2 LLIYSASFLYS SEQ ID NO: 266light chain ABR3 QQHYTTPP SEQ ID NO: 267 heavy chain ABR3 RWGGDGFYAMDVSEQ ID NO: 268 heavy chain ABR1 YSFTSYWIA SEQ ID NO: 269heavy chain ABR2 YMGLIYPGDSDTKY SEQ ID NO: 270 heavy chain ABR3RIIDVGYCSSSNCAKWPEYFQH SEQ ID NO: 271 light chain ABR1 SSNIGNNYVSSEQ ID NO: 272 light chain ABR2 LLIYGHTNRPA SEQ ID NO: 273light chain ABR3 AAWDDSLSGW SEQ ID NO: 274 heavy chain ABR1 YPFTNYGMNSEQ ID NO: 275 heavy chain ABR2 WMGWINTSTGESTF SEQ ID NO: 276heavy chain ABR3 RWEVYHGYVPY SEQ ID NO: 277 light chain ABR1 QDVYNAVASEQ ID NO: 278 light chain ABR2 LLIYSASSRYT SEQ ID NO: 279light chain ABR3 QQHFRTPF SEQ ID NO: 280 heavy chain ABR1 ITFSINTMGSEQ ID NO: 281 heavy chain ABR2 LVALISSIGDTYYA SEQ ID NO: 282heavy chain ABR3 KRFRTAAQGTDY SEQ ID NO: 283 heavy chain CDR1 GFNIKDTYIHSEQ ID NO: 284 heavy chain CDR2 RIYPTNGYTRYADSVKG SEQ ID NO: 285heavy chain CDR3 WGGDGFYAMDY SEQ ID NO: 286 light chain CDR1 RASQDVNTAVASEQ ID NO: 287 light chain CDR2 SASFLYS SEQ ID NO: 288 light chain CDR3QQHYTTPPT SEQ ID NO: 289 heavy chain CDR1 GFNIKDTYIH SEQ ID NO: 290heavy chain CDR2 RIYPTNGYTRYADSVKG SEQ ID NO: 291 heavy chain CDR3WGGDGFYAMDV SEQ ID NO: 292 light chain CDR1 RASQDVNTAVA SEQ ID NO: 293light chain CDR2 SASFLYS SEQ ID NO: 294 light chain CDR3 QQHYTTPPTSEQ ID NO: 295 heavy chain CDR1 GYSFTSYWIA SEQ ID NO: 296heavy chain CDR2 LIYPGDSDTKYSPSFQG SEQ ID NO: 297 heavy chain CDR3HDVGYCSSSNCAKWPEYFQH SEQ ID NO: 298 light chain CDR1 SGSSSNIGNNYVSSEQ ID NO: 299 light chain CDR2 GHTNRPA SEQ ID NO: 300 light chain CDR3AAWDDSLSGWV SEQ ID NO: 301 heavy chain CDR1 GITFSINTMG SEQ ID NO: 302heavy chain CDR2 LISSIGDTYYADSVKG SEQ ID NO: 303 heavy chain CDR3FRTAAQGTDY SEQ ID NO: 304 heavy chain ABR1 FTFSDSWIH SEQ ID NO: 305heavy chain ABR2 WVAWISPYGGSTYY SEQ ID NO: 306 heavy chain ABR3RRHWPGGFDY SEQ ID NO: 307 light chain ABR1 QDVSTAVA SEQ ID NO: 308light chain ABR2 LLIYSASFLYS SEQ ID NO: 309 light chain ABR3 QQYLYHPASEQ ID NO: 310 heavy chain ABR1 YTFTSYVMH SEQ ID NO: 311heavy chain ABR2 WIGYVNPFNDGTKY SEQ ID NO: 312 heavy chain ABR3 RQAWGYPSEQ ID NO: 313 light chain ABR1 ESVEYYGTSLVQ SEQ ID NO: 314light chain ABR2 LLIYAASSVDS SEQ ID NO: 315 light chain ABR3 QQSRRVPYSEQ ID NO: 316 heavy chain ABR1 YTFTSYDVH SEQ ID NO: 317heavy chain ABR2 WMGWLHADTGITKF SEQ ID NO: 318 heavy chain ABR3RERIQLWFDY SEQ ID NO: 319 light chain ABR1 QGISSWLA SEQ ID NO: 320light chain ABR2 SLIYAASSLQS SEQ ID NO: 321 light chain ABR3 QQYNSYPYSEQ ID NO: 322 heavy chain ABR1 DTFSTYAIS SEQ ID NO: 323heavy chain ABR2 WMGGIIPIFGKAHY SEQ ID NO: 324 heavy chain ABR3RKFHFVSGSPFGMDV SEQ ID NO: 325 light chain ABR1 QSVSSYLA SEQ ID NO: 326light chain ABR2 LLIYDASNRAT SEQ ID NO: 327 light chain ABR3 QQRSNWPSEQ ID NO: 328 heavy chain ABR1 FTFSSYIMM SEQ ID NO: 329heavy chain ABR2 WVSSIYPSGGITFY SEQ ID NO: 330 heavy chain ABR3RIKLGTVTTVDY SEQ ID NO: 331 light chain ABR1 SSDVGGYNYVS SEQ ID NO: 332light chain ABR2 LMIYDVSNRPS SEQ ID NO: 333 light chain ABR3 SSYTSSSTRSEQ ID NO: 334 heavy chain CDR1 GFNIKDYFLII SEQ ID NO: 335heavy chain CDR2 WINPDNGNTVYDPKFQG SEQ ID NO: 336 heavy chain CDR3RDYTYEKAALDY SEQ ID NO: 337 light chain CDR1 RASGNIYNYLA SEQ ID NO: 338light chain CDR2 DAKTLAD SEQ ID NO: 339 light chain CDR3 QHFWSLPFTSEQ ID NO: 340 SLT-1A-combo27 KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 341 SLT-1A-combo28KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 342 SLT-1A-combo29KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 343 SLT-1A-combo30KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 344 SLT-1A-combo31KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 345 SLT-1A-combo32KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 346 SLT-1A-combo33KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 347 SLT-1A-combo34KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 348 SLT-1A-combo35KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL AALSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 349 SLT-1A-combo36KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFVFTLEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 350 SLT-1A-combo37KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVNLVPMVATVGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 351 SLT-1A-combo38KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTNLVPMVATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 352 SLT-1A-combo39KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGI LGDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 353 SLT-1A-combo40KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHILRFSVA HKASAVAA SEQ ID NO: 354SLT-1A-combo41 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHARNLVP MVATVASAVAA SEQ ID NO: 355SLT-1A-base1 KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 356 SLT-1A-base2 andKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL SNA-base1LMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 357 SLT-1A-base3 andKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL SNA-base2LMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 358 SLT-1A-base4 andKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL SNA-base3LMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 359 SLT-1A-base5KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 360 SLT-1A-base6KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 361 SLT-1A-base7KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 362 SLT-1A-base8KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFVFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 363 SLT-1A-base9 andKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL SNA-base4LMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL AALSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVAR SEQ ID NO: 364 SLT-1Abase10KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVGILGFVFTLEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS GDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 365 SLT-1Abase11KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVNLVPMVATVGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 366 SLT-1Abase12KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTNLVPMVATVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVAR SEQ ID NO: 367 SLT-1Abase13KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGI LGDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASRVAR SEQ ID NO: 368 SLT-1Abase14KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHILRFSVA HKASRVAR SEQ ID NO: 369SLT-1Abase15 KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHARNLVP MVATVASRVAR SEQ ID NO: 370Shiga Toxin A KEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDVRGIDPEEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo101ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 371 Shiga Toxin AAEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDVRGIDPEEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo102ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 372 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLS combo103ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 373 Shiga Toxin AKEFILRFSVAHKYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDVRGIDPEEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo104ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 374 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLS combo105ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 375 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSNLVPMVATVDVRGIDPEEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLS combo106ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGALSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 376 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo107DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 377 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGILGFVFTLDVRGIDPEEGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo108DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 378 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo109DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 379 Shiga Toxin AKEFILDFITAKTYVDSLNVIRSAIGTPLQTISSGGTSLL Subunit EffectorMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNL PolypeptideYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSADS combo110SYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDD LSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 380 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo111DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 381 Shiga Toxin AMEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISIGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo112DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 382 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo113DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 383 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo114DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 384 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo115DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 385 Shiga Toxin AAEFTLDFITAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo116DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 386 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo117DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 387 Shiga Toxin AKEFTLDFSIAATYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo118DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 388 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo119DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 389 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo120DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 390 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo121DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 391 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNAFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo122DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 392 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo123DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 393 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLTAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo124DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 394 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA combo125DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 395 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFVVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA combo126DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 396 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGVDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA combo127DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 397 Shiga Toxin AMEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS Subunit EffectorLLMIDSGIGDNLFAVGILGVDFTLGRFNNLRLIVERN PolypeptideNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS combo128ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO : 398 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLEGRFNNLRLIVERN PolypeptideNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS combo129ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 399 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLEGRFNNLRLIVERN PolypeptideNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS combo130ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 400 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFVFTLEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo131ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 401 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFVFTLEGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo132ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 402 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo133DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 403 Shiga Toxin AAEFILDFIAAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo134DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 404 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo135DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 405 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo136DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 406 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo137DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 407 Shiga Toxin AMEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo138DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 408 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo139DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 409 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo140DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGALSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 410 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo141DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 411 Shiga Toxin AAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo142DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 412 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo143DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 413 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNATAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo144DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 414 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo145DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 415 Shiga Toxin AKEFTLDFIIAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo146DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 416 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo147DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 417 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit EffectorLMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo148DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 418 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA combo149DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 419 Shiga Toxin AAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo150DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 420 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo151DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 421 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo152DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 422 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo153DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 423 Shiga Toxin AMEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTS Subunit EffectorLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERN PolypeptideNLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLS combo154ADSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAA SEQ ID NO: 424 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNVFYRFADFSHVTFPGTTAVTLSA combo155DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 425 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo156DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 426 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo157DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 427 Shiga Toxin AAEFILDFSTAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo158DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 428 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo159DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 429 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTTAVTLSA combo160DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGALSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 430 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo161DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 431 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTNLVPMVA combo162TVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 432 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNAFYRFADFSHVTFPGTNLVPMVA combo163TVSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEAVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 433 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA combo164DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRGIL GDVFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 434 Shiga Toxin AAEFILDFSIAKTYVDSLNVIRSAIGTPLQTISSGGTSLL Subunit EffectorMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNL PolypeptideYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADS combo165SYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRGILGD VFTLSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAA SEQ ID NO: 435 Shiga Toxin AKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo166DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHILRFSVAHK ASAVAA SEQ ID NO: 436Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo167DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGALSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHILRFSVAHK ASAVAA SEQ ID NO: 437Shiga Toxin A KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo168DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHARNLVPMV ATVASAVAA SEQ ID NO: 438Shiga Toxin A MEFTLDFIIAKTYVDSLNVIRSAIGTPLQTISCGGTSL Subunit EffectorLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNN PolypeptideLYVTGFVNATNNAFYRFADFSHVTFPGTTAVTLSA combo169DSSYTTLQRVAGISRTGMQINRHSLTTSYLALMSHSGTSLTQSAARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVALTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHARNLVPMV ATVASAVAA SEQ ID NO: 439Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 1LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTTVSS SEQ ID NO: 440Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 2LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKILIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 441 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 3LMIDSGSGDNLFAVNIVPMVATVGRFNNLRLIVER NNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 442Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 4LMIDNLVPMVATVVDVRGIDPEEGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKILISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 443Cell-targeting QVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVH molcecule 5WVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATL TADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQI VLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAFDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSL TTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASRVAR SEQ ID NO 444Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 6LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMS CKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSA VYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTF GAGTKLELK SEQ ID NO: 445Cell-targeting QVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVH molecule 7WVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATL TADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQI VLSQSPTILSASPGEKVMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLELKEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSL TTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNC HHHASRVAR SEQ ID NO: 446Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 8LMIDSGIGDNLFAVDILGFDFYLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTFSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPQVQLQQPGAELVKPGASVKMS CKTSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLTADKSSSTVYMQLSSLTSEDSA VYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTF GAGTKLEIK SEQ ID NO: 447Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 9LMIDSGIGDNLAVDILGFDFTLGRFNNLRLIVERNN LYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKGGGGSQVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSED TAVYYCARDDIAALGPFDYWGQGTLVTVSSSEQ ID NO: 448 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 10 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTISYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRIQRGFRTTL DDLSGASYVMTAEDVDLILNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPG ASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELS SLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 449 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 11 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYRGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSGGGGSGGGGSGGGG SGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNE MFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYPWGQGTLVTVSA SEQ ID NO: 450 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 12LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTF GQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAIS WVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGS PEGMDVWGQGTTVTVSS SEQ ID NO: 451Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 13LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRIGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVAKILNSHHHASAVAAEFPKPSTPPGSSGGAPQVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGIEWMGGIIPIFGKAHYAQKFQGRVITITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSGGGGSG GGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ QRSNWPTFGQGTKVEIK SEQ ID NO: 452Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 14LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISC KASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGGSQIQLQQSGPEVVKP GASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSS LTSEDTAVYFCANYGNYWFAYWGQGTQVTVSASEQ ID NO: 453 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 15 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 454 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 16 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQSVNTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 455 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 17LMIDSGIGDNLFAVDILGFDFTLGRFNNLLIVERNNLYVTGFVNRINNVFYRFADFSFEVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLITSYLDLMSFISGTSLIQSVARAMLRFVTVTAEALRFRQIQRGFRITL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVMTQAAPSIPVTPGESVSISCRSSKSLILNSNIGNTYLYWFLQRPGGSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQ HLEYPFIFGAGTKLELKGSTSGSGKPGSGEGSEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQ KPGQGLENVIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFD YWGQGTTLTVSS SEQ ID NO: 456Cell-targeting KEFTLDFSTAKTYVDSENVIRSAIGTPLQTISSGGTSL molecule 18LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVNITAEDVDLTLNWGRLSSVLPDYRGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDIRVTISCRASQDISNYLAWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGGGLVKPGGSLKLSCAASGFAFSFYDNISWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVTVSA SEQ ID NO: 457 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGISL molecule 19LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRYTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID NO: 458Cell-targeting KEFTLDFSTAKTYVDSIAVIRSAIGTPLQTISSGGTSL molecule 20LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYMVTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCCTVSGFSLISYGVHWVRQSPGKGLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKM NSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSS SEQ ID NO: 459 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 21 LMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGITAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLFDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVT1TCRASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 460 Cell-targeting KEFTLDFSTAKWVDSLNVIRSAIGTPLQTISSGGTSLmolecule 22 LMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRFISLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPERAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSE DTAVYYCARERIQLWFDYWGQGTLVWSSSEQ ID NO: 461 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 23 LMIDSGIGDNLFAVDILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSC AASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQOYLYHPATFGQGTKVEIKSEQ ID NO: 462 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 24 LMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRFTISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 463 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 25 LMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGNMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTFFCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSE DTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO 464 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 26 LMIDSGIGDNLFAVGILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSFTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRFSFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSC AASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTA VYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 465 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 27 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEFKGGGGSQVQLQESGPGLVRPSQTLSL FCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO 466 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 28 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGODSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSSA SEQ ID NO: 467Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 29LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVFRNN LYVTGFVNRTNNVFYRFADFSHVTFGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSH DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVES GGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKN TAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS SEQ ID NO: 468 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 30LMIDSGIGDNITAVDILGFDFMGRFNNLRLIVERNNLYVIGFVNRINNVFYRFADFSHVTFPCITTAVTLSA DSSYTYLQRVAGISRTGMQINIMSLTBYLDLMSHSATSLIQSVARAMLRFVTVTAEALRFRQIQRGFRITL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYIIGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIELIQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNAPPLLISGATSLEMVPSRFSGSGSGKDYTLSTISLQTEDVATYYCQQYAVSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVFMNRQSPGKGLEW LGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTV SS SEQ ID NO: 469 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 31LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGITAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTYTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARFYPTNGYTRYADSVKGRF FISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 470 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 32LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTVLYWFLQRPGQSPQLLIYRMSNLA SGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQL QQSGPELIKPGASVKMSCKASGYTFTSYVMKWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDK SSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSS SEQ ID NO: 471 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 33LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGINNWINNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRITL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISCRASQDISNYLAWYQQKPDGIVKLLFYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWIFGCGTKLEIKGSTSGSGKPGSGEGSEVQLVESGG GLVKPGGSLKLSCAASGFAFSFYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMNYCARHSGYGTHWGVLFAYWGQGTLVTVSA SEQ ID NO: 472 Cell-targetingKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 34LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSNAILGSVALAINSHHHASAVAAEFPKPSIPPGSSGGAPDIVLTQSPASIAVSLGQRATISC KASQSVDFDGDSYMNWYQQKPGQPPKVLWAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGGSQIQLQQSGPEVVKP GASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKNNEKFKGKATLTVDTSSSTAFMQLSS LTSEDTAVYFCANYGNYWFAYWGQGTQVTVSASEQ ID NO: 473 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 35 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTIAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYFIGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQITLKESGPGILQPSQTLSLTCSFSGFSLTFYGIGVGWIRQPPGKGLEWLTHIWWNDNK YYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSAGGGGSDVVMTQTPLS LPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDLGVYFCSQSTHVPWTFGGGTKLEIKSEQ ID NO: 474 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 36 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPG ASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELS SLTSEDSAVYYGARQAWGYPWGQGTLVTVSASEQ ID NO: 475 Cell-targeting KEFTLDFSTAKTVVDSLNVIRSAIGTPLQTISSGGTSLmolecule 37 LMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRFISLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTYVYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYITGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSTLVTVSSSEQ ID NO: 476 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 38 LMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSE DTAVYYCARERIQLWFDYWGQGTLVTVSSSEQ ID NO: 477 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 39 LMIDSGIGDNLFAVGILGFVFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPEVQLVESGGGLVQPGGSLRLSC AASGFTFSDSWFHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSERAEDTA VYYCARRHWPGGFDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKLGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQYLYHPATFGQGTKVEIKSEQ ID NO: 478 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 40 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTTFCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO 479 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 41 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAIGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQUYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFNIKDTYIHWYRQAPGKGLEWVARIYPFNGYTRYADSVKGRFTISADISKNTAYLQMNSLRA EDTAVYYCSRWGGDGFYAMPYWGQGTLVTVSSASEQ ID NO: 480 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 42 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFWRTNNVFYRFADFSHVTFPGTTAVTLSG DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRGIL GDVFTRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNOVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID NO: 481Cell-targeting KEFTLDFSTAKTYVDSENVIRSAIGTPLQTISSGGTSL molecule 43LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTMAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHS ATSLTQSVARAMLFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYFIGQ DSVRVGRISFGSINAILGSVALILNSHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSNSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID NO: 482Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 44LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVIFPGITAVTLSA DSSYTFLQRVAGISRTGMQINRHSLTISYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMPSWGQGSLVTVSSSEQ ID NO: 483 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSCiGTSLmolecule 45 LMIDSGIGDNLFAVDELGFDFTLGRFNNLRIIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAIYPTNGYTRYADSVKGRF EISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMPYWGQGTLVTVSS SEQ ID NO: 484 Cell-targetingAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 46LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAFDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLFAVIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMnTGFVMDSWGQGSLVTVSSSEQ ID NO: 485 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 47 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSITISATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLILNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWTQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTCFVMDSWGQGSLVTVSSSEQ ID NO: 486 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 48 LMIDSGGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAITYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA EDTAVYYCSRWGGPGFYAMDYWGQGTLVTVSSASEQ ID NO: 487 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 49 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFIRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLITSYLDLMSHSATSITQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYEGQDSVRVGTISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITC KASQSVDFDGDSYMNWYQQKPGQPPKVILYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLHKGGGGSQIQLQQSGPEVVKP GASVKISCKASGYTFIDYYFFWVKQKPGQGLEWIGWIYPGSGNIKYNEKFKGIKATLTVDTSSSTAFMQLSS LTSEDTAVYFCANYGNYWFAYINGQGTQVTVSASEQ ID NO: 488 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 50 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLA SGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKGSTSGSGKPGSGEGSEVQL QQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDK SSSTAYMELSSLTSEDSAVYYCARGTYYYGSRVFDYWGQGTTLTVSS SEQ ID NO: 489 Cell-targetingQVQLQQPGAELVKPGASVKMSCKTSGYTFTSYNVH molecule 51WVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATL TADKSSSTVMQLSSLTSEDSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGSQI VLSQSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPPTFGAGTKLFLKEFPKPSTPPGSSGGAPKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRHSLT TSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSH HHASAVAA SEQ ID NO: 490Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 52LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQTTSSLSASLGDRVTISCRASQDISNYLAWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIKGSTSGSGKPGSGECSEVQLVESGG GLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLY LQMSSLKSEDTAMYYCARHSGYGTFIWGVLFAYWGQGTLVTVSA SEQ ID NO: 491 Cell-targetingKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 53LMIDSGIGDNLFAVDELGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGR GLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQ GSLVTVSS SEQ ID NO: 492Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 54LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINIAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWTQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPTFGGGTKLEIKGSTSGSGKPGSGEGSKVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQAPGKGLEW LGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDTAIYFCAKTLITTGYAMDYWGQGTTVTV SS SEQ ID NO: 493 Cell-targetingKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 55LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERMNLYVTCFVNRTNNVFYOTADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPQITLKESGPGILQPSQTLSLTCSFSGFSLTTYGIGVGWTRQPPGKGLEWLTHIWWNDNK YYNTALRSRLTISKDSSNNQVLLKIANVDTADTATYYCLYGYTYWGQGTLVTVSAGGGGSDVVMTQTPLS LPVSLGDQASISCRSSQSLLYSNGNTYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRV EAEDLGVYFCSQSTHVPWTFGGGTKLEIKSEQ ID NO: 494 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 56 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEAERFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 495 Cell-targetingKEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 57LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIKGGGGSEVQLQQSGPELVKPG ASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYVNPFNDGTKYNEMFKGKATLTSDKSSSTAYMELS SLTSEDSAVYYCARQAWGYPWGQGTLVTVSASEQ ID NO: 496 Cell-targeting AEFILFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 58 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 497 Cell-targeting AEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 59 LMIDSGIGDNLFAVNLVPMVATVGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYMGQDSVRVGRISFGSINAILGSVAULNSHHHASAVAAEFPKRSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVROPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSERESSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 498 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 60 LMIDSGIGDNLFAVDVRGIAPIEARFNNLREIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL AALSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDLATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 499 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 61 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHARNLVPMVATVASAVAAEFPKPSTPPGSSGGAPDFQMTQSPSSLSASVGDRVTITCKASEDIYNRLIWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDLATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGP GLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVS LRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSS SEQ ID NO: 500 Cell-targetingKEFTLDSFSTAKTYVDSLNVISAIGTPLQTISSGGTSL molecule 62LMIDSGSGDNLFAVDILGFDFTLGRFNNLRLIVERN NLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLS LTCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAA DTAVYYCAKSMITTGFVMDSWGQGSLVTVSSSEQ ID NO: 501 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 63 LMIDSGSGDNLFAVDVRGIAPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLS ADSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTT LDDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFSLTSYGVHWVRQPPG RGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAADTAVYYCAKSMITTGFVMDSWG QGSLVTVSSA SEQ ID NO: 502Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 64LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDVQVTQSPSSLSASVGDRVTITCRSSQSIANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCLQGTHQPYTFGQGTKVEIKGGGGSEVQLVQSGAEVKKP GASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWIGGINPGNNYATYRRKFQGRVTMTADTSTSTVYMEL SSLRSEDTAVYYCTREGYGNYGAWFAYWGQGTLVTVSSA SEQ ID NO: 503 Cell-targetingAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 65LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTYTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIVLTQSPASEAVSLGQRATISC KASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIKGGGGSQIQLQQSGPEVVKP GASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSS LTSEDTAVYFCANYGNYWFAYWGQGTQVTVSASEQ ID NO: 504 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 66 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRIGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTALDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLL1SGATSLETGVPSRFSGSGSGTDFTFTTSSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSASEQ ID NO: 505 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 67 LMIDSGIGDNLFAVDVRGIAPIEARFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSADSSYTTLQRVAGISRTGMQINRFISLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL AALSGASYVMTEDVDLTLNWGRLSSVLPDYHGQDVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVnTCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSIDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMPSWGQGSLVTVSSASEQ ID NO: 506 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 68 LMIDSGIGDNLFAVDELGFDFILGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTLTCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGQGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNFTKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSASEQ ID NO: 507 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 69 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQFNRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSASEQ ID NO: 508 Cell-targeting AEFILDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 70 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVnTCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRLNTIKDNSKNQVSLRLSSVTAAD TAVYYCAKSMHTGFVMDSWGQGSLVTVSSASEQ ID NO: 509 Cell-targeting KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 71 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGODSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDFFNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTFSSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYMAAFMSRLNITKDNSKNQVSLRLSSVIAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSASEQ ID NO: 510 Cell-targeting KEFILDFSTAKTYVDSLNVIRSAIGTPLQTTSSGGTSLmolecule 73 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGASYVMTAEDVDLTLNWGRLSSVEPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGGGGSQVQLQESGPGLVRPSQTLSL TCTVSGFSLTSYGVHWVRQPPGRGLEVVIGVMWRGGSTDYNAAFMSRLNITKDNSKNQVSLRLSSVTAAD TAVYYCAKSMITTGFVMDSWGQGSLVTVSSASEQ ID NO: 511 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 74 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSEQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA EDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASEQ ID NO: 512 Cell-targeting AEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLmolecule 75 LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGQPAIQMSQSPASLSASVGETVTITC RASGNIYNYLAWYQQKQGKSPHLLVYDAKTLADGVPSRFSGSGSGTQYSLKISSLQTEDSGNYYCQHFWSLPFTFGSGTKLEIKGSTSGSGKPGSGEGSTGEVQLQ QSGAELVRPGALVKLSCKTSGFNIKDYFLHWVRQRPDQGLEWIGWINPDNGNTVYDPKFQGTASLTADTS SNTVYLQLSGLTSEDTAVYFCTRRDYTYEKAALDYWGQGTTVTVST SEQ ID NO: 513 Cell-targetingAEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSL molecule 76LMIDSGIGDNLFAVDILGFDFTLGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSA DSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSATSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTL DDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNSHHHASAVAAEFPKPSTPPGSSGGAPDIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIKGSTSGSGKPGSGEGSTKGQVQ LQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGEPTFDDDFKGRFAFSLD TSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTLVTVSS

The invention is claimed as follows:
 1. A cell-targeting moleculecomprising: i) a binding region capable of specifically binding anextracellular target biomolecule physically coupled to the cellularsurface of a cell, wherein the extracellular target biomolecule isHER2/neu/ErbB2, PDL1, CD38, CD20, BCMA, SLAMF7, or CTLA-4, and ii) aShiga toxin effector polypeptide comprising an amino acid sequencehaving at least 90% identity to amino acids 1 to 251 of SEQ ID NO: 1,wherein the amino acid sequence comprises the amino acid substitutions:S45I, V54I, R55L, I57F, P59F, E60T, E61L, G110A, R188A, C242S, R248A andR251A according to SEQ ID NO: 1; and wherein the amino acid sequencecomprises an asparagine at the amino acid residue corresponding toposition 75 of SEQ ID NO: 1, a tyrosine at the amino acid residuecorresponding to position 77 of SEQ ID NO: 1, a tyrosine at the aminoacid residue corresponding to position 114 of SEQ ID NO: 1, a glutamateat the amino acid residue corresponding to position 167 of SEQ ID NO: 1,an arginine at the amino acid residue corresponding to position 170 ofSEQ ID NO: 1, an arginine at the amino acid residue corresponding toposition 176 of SEQ ID NO: 1, and a tryptophan at the amino acid residuecorresponding to position 203 of SEQ ID NO:
 1. 2. The cell-targetingmolecule of claim 1, wherein the Shiga toxin effector polypeptidecomprises or consists of an amino acid sequence having at least 95%sequence identity to amino acids 1 to 251 of SEQ ID NO:
 1. 3. Thecell-targeting molecule of claim 1, wherein the Shiga toxin effectorpolypeptide comprises or consists of an amino acid sequence having atleast 95% sequence identity to SEQ ID NO:
 13. 4. The cell-targetingmolecule of claim 1, wherein the binding region is fused to the carboxyterminus of the Shiga toxin effector polypeptide to form a single,continuous polypeptide.
 5. The cell-targeting molecule of claim 1,wherein the binding region comprises at least one immunoglobulin-typebinding region.
 6. The cell-targeting molecule of claim 5, wherein theat least one immunoglobulin-type binding region comprises a polypeptideselected from: single-domain antibody fragment, single-chain variablefragment, antibody variable fragment, complementary determining region 3fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment,antigen-binding fragment, fibronectin-derived 10th fibronectin type IIIdomain, tenascin type III domain, ankyrin repeat motif domain,low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitzdomain, Protein-A-derived Z domain, gamma-B crystallin-derived domain,ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2domain, miniprotein, C-type lectin-like domain scaffold, a heavy-chainantibody domain derived from a camelid V_(H)H fragment, heavy-chainantibody domain derived from cartilaginous fish, immunoglobulin newantigen receptor (IgNAR), V_(NAR) fragment, multimerizing scFv fragment,bivalent minibody, bispecific tandem scFv, bispecific tandem V_(H)H, andbispecific minibody.
 7. The cell-targeting molecule of claim 6, whereinthe multimerizing scFv fragment comprises a diabody, a triabody, or atetrabody.
 8. The cell-targeting molecule of claim 1, which comprisesthe linker peptide shown in any one of SEQ ID NO: 540-543, 544-550, and553-559.
 9. The cell-targeting molecule of claim 1, which comprises thelinker peptide shown in any one of SEQ ID NO: 540-543 and 553-557.
 10. Apharmaceutical composition comprising the cell-targeting molecule ofclaim 1 and a pharmaceutically acceptable excipient or carrier.
 11. Apolynucleotide encoding the cell-targeting molecule of claim 1, or acomplement thereof.
 12. An expression vector comprising thepolynucleotide of claim
 11. 13. A host cell comprising thepolynucleotide of claim
 11. 14. A host cell comprising the expressionvector of claim
 12. 15. A method of treating a disease, disorder, orcondition in a patient in need thereof, comprising administering to thepatient a therapeutically effective amount of the cell-targetingmolecule of claim 1 or a pharmaceutical composition thereof.
 16. Themethod of claim 15, wherein treating the disease, disorder or conditioncomprises selectively killing target cells of the disease, disorder orcondition, and wherein the target cells express the extracellular targetbiomolecule.
 17. The method of claim 15, wherein the disease, disorder,or condition is a cancer, immune disorder, or microbial infection. 18.The method of claim 17, wherein the cancer is bone cancer, breastcancer, central/peripheral nervous system cancer, gastrointestinalcancer, germ cell cancer, glandular cancer, head-neck cancer,hematological cancer, kidney-urinary tract cancer, liver cancer,lung/pleura cancer, prostate cancer, sarcoma, skin cancer, or uterinecancer.
 19. The method of claim 15, wherein said administering is viaintravenous administration.